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Universidad de Concepción
Dirección de Postgrado Facultad de Agronomía - Programa de Doctorado en Ciencias Agropecuarias
Detección y tipificación molecular de Staphylococcus aureus y Staphylococcus aureus resistente a meticilina
(SARM) en Dakota del Norte, Estados Unidos
Detection and molecular typing of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) in
North Dakota, United States
VALERIA CRISTINA VELASCO PIZARRO CHILLÁN-CHILE
2014
Profesor Guía: Pedro Rojas García Dpto. de Ciencias Pecuarias, Facultad de Ciencias Veterinarias
Universidad de Concepción
Detección y tipificación molecular de Staphylococcus aureus y Staphylococcus aureus resistente a meticilina (SARM) en Dakota del Norte,
Estados Unidos
Aprobada por: Prof. Pedro Rojas García ________________________ Médico Veterinario, Dr. Med. Vet. Profesor Guía Prof. Álvaro Ruiz Garrido ________________________ Médico Veterinario, Ph. D. Evaluador Interno Prof. Cristián Balbontín Sepúlveda ________________________ Ing. Agrónomo, Dr. Cs. Evaluador Interno Prof. Heriberto Fernández Jaramillo ________________________ Tecnólogo Médico, Dr. Cs. Evaluador Externo Prof. Manuel Quezada Orellana ________________________ Médico Veterinario, Dr. Med. Vet. Director de Programa
RECONOCIMIENTO
Agradezco el financiamiento otorgado por North Dakota State University (Fargo, ND, USA)
para el desarrollo de esta investigación, y a la Universidad de Concepción por apoyarme y
otorgarme la beca funcionario.
A mi profesor guía Dr. Pedro Rojas García, por su apoyo en esta tesis, por sus valiosos
consejos y su sincera amistad.
A mi profesora guía externa Dr. Catherine M. Logue, por su preocupación, entusiasmo,
energía y sencillez. Por creer en mí y ser un ejemplo para mí.
A mi familia, que me apoya en forma incondicional en cada desafío y me acompaña siempre.
A mis amigos en Chile y alrededor del mundo, gracias por alentarme a la distancia y por los
buenos momentos. A todos ellos, gracias por su tolerancia y respeto.
Y a la fuerza divina que recibo cada día.
PRÓLOGO
Esta investigación fue desarrollada en North Dakota State University, Fargo, Dakota del Norte,
Estados Unidos durante los años 2010 y 2011.
El escrito de esta tesis consta de una parte general en idioma español (Introducción General,
Discusión General y Conclusiones Generales) y tres capítulos en inglés (Desarrollo 1., 2. y 3.)
que corresponden a tres artículos científicos. El formato de cada capítulo corresponde al
formato exigido por cada revista. La numeración de tablas y figuras se inicia en cada capítulo
para mantener el orden de citación dentro de cada artículo.
Las abreviaciones en español e inglés que aparecen en el texto se encuentran en el Glosario de
Abreviaciones.
ÍNDICE
RESUMEN GENERAL ...............................................................................................................
GENERAL ABSTRACT .............................................................................................................
I. INTRODUCCIÓN GENERAL ...............................................................................................
1. Características de Staphylococcus aureus..............................................................................
2. Mecanismos de resistencia a meticilina.................................................................................
3. Tipificación molecular de Staphylococcus aureus y SARM.................................................
4. Prevalencia de Staphylococcus aureus y SARM...................................................................
5. Hipótesis y objetivos..............................................................................................................
6. Referencias.............................................................................................................................
II. DESARROLLO
1. Molecular typing of Staphylococcus aureus and methicillin-resistant S. aureus
(MRSA) isolated from animals and retail meat in North Dakota, United States...............
1.1 Introduction..........................................................................................................................
1.2 Materials and methods.........................................................................................................
1.2.1 Samples.......................................................................................................................
1.2.2 Isolation of S. aureus and MRSA...............................................................................
1.2.3 Multiplex polymerase chain reaction (PCR)...............................................................
1.2.4 PFGE...........................................................................................................................
1.2.5 Multilocus sequence typing (MLST)..........................................................................
1.2.6 Antimicrobial susceptibility testing............................................................................
1.2.7 Statistical analysis.......................................................................................................
1.3 Results..................................................................................................................................
1.4 Discussion............................................................................................................................
1.5 Conclusion...........................................................................................................................
1.6 References...........................................................................................................................
2. Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-
Valentine Leukocidin (PVL) genes from selective enrichments from animals and retail
meat...........................................................................................................................................
2.1 Introduction..........................................................................................................................
2.2 Materials and methods.........................................................................................................
1
5
9
10
11
13
15
18
19
27
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52
54
2.2.1 Samples.......................................................................................................................
2.2.2 Culture method............................................................................................................
2.2.3 Conventional multiplex PCR method.........................................................................
2.2.4 Multiplex real-time PCR assay...................................................................................
2.2.5 Characterization of S. aureus strains isolated by culture method...............................
2.2.6 Statistical analysis.......................................................................................................
2.3 Results..................................................................................................................................
2.4 Discussion............................................................................................................................
2.5 References............................................................................................................................
3. Staphylococcus aureus harboring mecA gene and genes encoding Panton-Valentine
Leukocidin (PVL) exotoxin from humans.............................................................................
3.1 Introduction..........................................................................................................................
3.2 Materials and methods.........................................................................................................
3.2.1 Samples.......................................................................................................................
3.2.2 Multiplex polymerase chain reaction (mPCR)............................................................
3.2.3 Statistical analysis.......................................................................................................
3.3 Results..................................................................................................................................
3.4 Discussion............................................................................................................................
3.5 Conclusion...........................................................................................................................
3.6 References...........................................................................................................................
III. DISCUSIÓN GENERAL ......................................................................................................
IV. CONCLUSIONES GENERALES ........................................................................................
V. REFERENCIAS......................................................................................................................
54
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ÍNDICE DE TABLAS
Table 1 (Desarrollo 1)
Table 2 (Desarrollo 1)
Table 3 (Desarrollo 1)
Table 4 (Desarrollo 1)
Table 5 (Desarrollo 1)
Table 1 (Desarrollo 2)
Table 2 (Desarrollo 2)
Table 3 (Desarrollo 2)
Table 4 (Desarrollo 2)
Nucleotide sequence of the primers used in multiplex polymerase
chain reaction for detection of 16S rRNA, mecA, and Panton-
Valentine leukocidin genes (McClure et al., 2006); and multilocus
sequence typing analysis for detection of arcC, aroE, glpF, gmk, pta,
tpi, and yqiL genes (Enright et al., 2000)...............................................
Identification of 16S rRNA, mecA and Panton-Valentine leukpcidin
(PVL) genes in Staphylococcus aureus and methicillin-resistant
Staphylococcus aureus isolates from animals and retail meat...............
Antimicrobial resistance of Staphylococcus aureus and methicillin-
resistant S. aureus (MRSA) isolates from animals and retail meat........
Minimum inhibitory concentrations (MICs) of resistant
Staphylococcus aureus and methicillin-resistant S. aureus isolates
from animals and retail meat..................................................................
Antimicrobial resistance profiles of Staphylococcus aureus and
methicillin-resistant S. aureus (MRSA) isolates from animals and
retail meat...............................................................................................
Nucleotide sequence of the primers and probes used in conventional
multiplex PCR, multiplex real-time PCR...............................................
Detection of S. aureus, mecA and PVL genes from animals and retail
meat using a conventional culture/PCR method and a real-time PCR
assay.......................................................................................................
Raw agreement indices among conventional culture/PCR method and
real-time PCR assay, with two-step enrichment procedure for
detection of S. aureus from animals and retail meat..............................
Raw agreement indices among conventional culture/PCR method and
real-time PCR assay, with two-step enrichment procedure for
detection of mecA gene from animals and retail meat...........................
44
45
46
47
48
68
69
70
71
Table 5 (Desarrollo 2)
Table 1 (Desarrollo 3)
Table 2 (Desarrollo 3)
Antimicrobial resistance profiles and sequence types of S. aureus
isolated by conventional culture/PCR method from animals and retail
meat........................................................................................................
Nucleotide sequence of the primers used in the multiplex PCR for
detection of the 16S rRNA gene, the mecAgene and the PVL genes
(McClure et al., 2006)............................................................................
Identification of 16S rRNA, mecA and Panton-Valentine Leukocidin
(PVL) genes in Staphylococcus aureus isolates from healthy people
and methicillin-resistance Staphylococcus aureus (MRSA) isolates
from hospital patients.............................................................................
72
84
85
ÍNDICE DE FIGURAS
Figura 1 (Resumen
General)
Prevalencia de Staphylococcus aureus en animales, carne cruda y
productos cárnicos..................................................................................
3
Figure 2 (General
Abstract)
Prevalence of Staphylococcus aureus in animals, raw meat, and deli
meat........................................................................................................
7
Figure 1 (Desarrollo 1) Dendrogram showing the genetic relatedness of 100 Staphylococcus
aureus isolates as determined by the analysis of pulsed-field gel
electrophoresis (PFGE) profiles by the unweighted-pair group method
with arithmetic averages with the sequence type (ST). The scale
indicates levels of similarity within this set of isolates. Red line
indicates the cutoff at 80%. Numbers represent the samples codes,
followed on the right by the STs and the type of the sample. Clusters
are separated by horizontal lines and labeled with correlative
numbers. Asterisks indicate methicillin-resistant Staphylococcus
aureus isolates determined by multiplex polymerase chain reaction
targeting the mecA gene.........................................................................
49
GLOSARIO DE ABREVIACIONES
AR: Antimicrobial-resistant
BP: Baird Parker
CA-MRSA: Community-associated MRSA
CDC: Centers for Disease Control and Prevention
CHL: Chloramphenicol
CIP: Ciprofloxacin
ERY: Erythromycin
FDA: Food and Drug Administration
GEN: Gentamicin
GPID: Gram positive identification
HA-MRSA: Health care-associated MRSA
KAN: Kanamycin
LA-MRSA: Livestock-associated MRSA
LINC: Lincomycin
MDR: Multidrug resistance (resistant)
MHB: Mueller-Hinton broth
MIC: Minimum inhibitory concentration
MLST: Multilocus sequence typing
mPCR: Multiplex PCR
MR: Multirresistente (resistencia a múltiples agentes antimicrobianos)
MRSA: Methicilin-resistant S. aureus
MSSA: Methicillin-susceptible S. aureus
NARMS: National Antimicrobial Resistance Monitoring System
QUI/DAL: Quinupristin/dalfopristin
PBP2a: Proteína de unión a la penicilina alterada
PCR: Polymerase chain reaction
PCRm: PCR múltiple
PEN: Penicillin
PFGE: Pulsed-field gel electrophoresis
PHMB+: Phenol red mannitol broth + ceftizoxime + aztreonam
PVL: Leucocidina de Panton-Valentine
SARM: Staphylococcus aureus resistente a meticilina
SB: Sheep blood
SCCmec: Cassette cromosómico estafilocócico mec
ST: Serotipo
STR: Streptomycin
TET: tetracycline
TSA: Trypticase soy agar
USDA: U.S. Department of Agriculture
1
Detección y tipificación molecular de Staphylococcus aureus y
Staphylococcus aureus resistente a meticilina (SARM) en Dakota del Norte,
Estados Unidos.
Detection and molecular typing of Staphylococcus aureus and methicillin-
resistant Staphylococcus aureus (MRSA) in North Dakota, United States.
RESUMEN GENERAL
La emergencia y la propagación de patógenos resistentes a agentes antimicrobianos ha
aumentado la preocupación en materia de salud pública a nivel mundial. El uso de antibióticos
en producción animal, en profilaxis, terapia y promoción del crecimiento, se asocia a la
emergencia de patógenos resistentes en el ganado. La exposición del ganado a dosis
subterapéuticas de agentes antimicrobianos genera un reservorio de patógenos resistentes en
los animales con el riesgo de transmisión a las personas a través de la cadena productiva de la
carne. Uno de los patógenos que desarrolla rápidamente resistencia a múltiples agentes
antimicrobianos (MR) es Staphylococcus aureus. En especial, las cepas de S. aureus resistente
a meticilina (SARM) constituyen una causa importante de infecciones asociadas a hospitales
(HA-MRSA), a la comunidad (CA-MRSA) y al ganado (LA-MRSA). La capacidad de
colonizar las fosas nasales y piel de humanos y animales que posee S. aureus y la detección de
cepas de SARM y S. aureus MR en animales de abasto y en carne, aumentan la probabiliadad
de contaminación de los alimentos en las diferentes etapas de la cadena productiva. La
resistencia a la meticilina se atribuye a la proteína de unión a la penicilina 2a (PBP2a)
codificada en el gen mecA, que forma parte del casssette cromosómico estafilocócico mec
(SCCmec). Esta proteína le confiere a las cepas de SARM una baja afinidad por antibióticos β-
lactámicos. A su vez, las cepas CA-MRSA produccen generalmente la exotoxina Leucocidina
de Panton-Valentine (PVL), la cual constituye un factor de virulencia asociado a infecciones
de la piel y necrosis de tejidos. Por lo tanto, debido al impacto que pueden tener estas cepas de
S. aureus en la salud humana, resulta relevante evaluar si los productos alimenticios pueden
ser un foco de contaminación que facilite la propagación de estas infecciones.
2
El objetivo de este estudio fue establecer la prevalencia y los tipos moleculares de S.
aureus y SARM en animales de abasto, carne cruda, productos cárnicos y humanos,
determinando la relación filogenética entre las cepas.
Se aisló S. aureus desde 167 muestras nasales de animales, 145 muestras de carne
cruda y 46 muestras de productos cárnicos, utilizando una etapa de enriquecimiento selectivo
en dos pasos, seguido de cultivo en placa. Los aislados que resultaron ser positivos se
sometieron al análisis por PCR múltiple (PCRm) para identificar los genes: ARNr 16S
(identificación de S. aureus), mecA (detección de SARM) y PVL (factor de virulencia
asociado a CA-MRSA). Para la tipificación molecular de las cepas de S. aureus se utilizó
electroforesis en gel de campo pulsado (PFGE) y tipificación de secuencias multilocus
(MLST). El análisis de susceptibilidad antimicrobiana se llevó a cabo a través del método de
microdilución en caldo. Además, se aisló e identificó S. aureus desde muestras de fosas
nasales de 550 personas sanas a través de métodos de cultivo y análisis bioquímico. Un total
de 108 aislados clínicos de cepas SARM obtenidos de pacientes hospitalizados se analizaron a
través de PCRm para confirmar la presencia de S. aureus y detectar las cepas con los genes
mecA y PVL. Se desarrolló un método de PCR múltiple en tiempo real con el fin de disminuir
el tiempo de análisis de muestras de animales y carne y para aumentar la sensibilidad de
detección de S. aureus y de los genes mecA y PVL. Este procedimiento se realizó con
posterioridad al enriquecimiento primario y al secundario que preceden al cultivo en placa.
Los resultados se compararon con el método de cultivo que incorpora el enriquecimiento
selectivo en dos pasos para aislar S. aureus. Se analizó un total de 234 muestras a través de
estos métodos (77 muestras nasales de animales, 112 muestras de carne cruda y 45 muestras
de productos cárnicos).
La prevalencia total de S. aureus determinada luego del análisis de las muestras por el
método de cultivo con doble enriquecimiento fue de 34,7% en animales, siendo más alta en
cerdos (50,0%) y corderos (40,6%) (P<0,05). En carne cruda la prevalencia fue de 47,6%,
siendo más alta en carne de pollo (67,6%) y de cerdo (49,3%) (P<0,05). En productos cárnicos
la prevalencia fue de 13,0% (Figura 1). Cinco muestras de carne de cerdo (7,0%) fueron
positivas para el gen mecA, de las cuales tres correspondieron al serotipo ST398 y dos al
serotipo ST5. Todas estas cepas exhibieron resistencia a la penicilina y cuatro fueron MR. En
ninguna de las muestras se detectaron a través de PCRm los genes PVL. Los serotipos más
3
comunes en corderos fueron ST398 y ST133, en cerdos y carne de cerdo ST398 y ST9 y en
carne de pollo ST5. Se determinó una similitud genética entre las cepas de S. aureus ST9
aisladas de cerdos y carne de cerdo. La mayoría de las cepas de S. aureus susceptibles a
agentes antimicrobianos fueron ST5 aisladas de carne de pollo. Los aislados MR fueron
encontrados en cerdos, carne de cerdo y corderos.
0
10
20
30
40
50
60
70
80
Pre
vale
nci
a (%
)
Corderos
Cerdos
Vacunos
Cerdo
Pollo
Vacuno
Jamón de cerdo
Jamón de pavo
Jamón de pollo
Figura 1. Prevalencia de Staphylococcus aureus en animales, carne cruda y productos cárnicos.
La proporción de personas portadoras sanas de S. aureus en las fosas nasales fue de
7,6%. Ninguna de estas cepas fue positiva para los genes mecA y PVL. Un total de 105 cepas
de SARM (97,2%) obtenidas de pacientes hospitalizados presentaron el gen mecA y 11
(10,2%) los genes PVL.
La aplicación del método de PCR en tiempo real en muestras con enriquecimiento
primario y secundario permitió detectar S. aureus en 111/234 y 120/234 muestras,
respectivamente. Estos niveles de detección fueron mayores que los obtenidos con el método
de cultivo, el cual permitió detectar 95/234. Para la detección de S. aureus a través de PCR en
tiempo real, la concordancia total con el método de cultivo fue de 83,9-97,8% (kappa=0,68-
0,88 [considerable a casi perfecta]) y 68,9-88,3% (kappa=0,29-0,77 [pobre a considerable])
para el enriquecimiento primario y secundario, respectivamente. Para la detección del gen
Animales Carne Prod. Cárnicos
4
mecA, la concordancia total fue de 91,1-98,7% y 86,7-98,7% (kappa=0-0,49 [no concordancia
más allá del azar a moderada]) para el enriquecimiento primario y secundario, respectivamente.
El método de PCR en tiempo real detectó el gene mecA en algunas muestras que fueron
negativas para S. aureus, pero positivas para Staphylococcus spp.
La presencia de SARM, S. aureus MR, y el serotipo ST398 en la cadena productiva de
la carne indica que existe un riesgo de transmisión a las personas. La similitud genética
encontrada entre las cepas de origen porcino (animales y carne) sugiere una posible
contaminación de la carne durante la faena. Cepas de S. aureus que presentan los genes mecA
y PVL se encuentraron en pacientes con infecciones invasivas. La proporción de portadores
sanos de S. aureus es baja, sin presentar los genes mecA y PVL, lo cual representa un riesgo
menor de transmisión a la comunidad. El método de PCR en tiempo real puede recomendarse
como una técnica rápida para la detección de S. aureus y el gen mecA, con la posterior
confirmación de SARM a través del método estándar de cultivo.
Los resultados de este trabajo sugieren que debe implementarse una vigilancia efectiva
en la cadena productiva de la carne con el fin de monitorear SARM y S. aureus MR y
establecer acciones para disminuir la propagación de cepas resistentes a antibióticos.
Palabras claves: Staphylococcus aureus resistente a meticilina (SARM), multirresistente (MR),
mecA, Leucocidina de Panton- Valentine (PVL), carne cruda, animales, productos cárnicos,
aislados clínicos, personas sanas.
5
GENERAL ABSTRACT
The emergence and the spread of antimicrobial-resistant (AR) pathogens has increased
the public health concern worldwide. The emergence of AR pathogens in food-producing
animals has been asociated with the use of antibiotics in animal production in prophylaxis,
therapy, and growth promotion. The exposure to sub-therapy doses of antimicrobial agents has
created a reservoir of AR pathogens in animals with the risk of transmission to humans
through the meat production chain. One of the pathogens that rapidly develop multidrug
resistance (MDR) is Staphylococcus aureus. Particularly, methicillin-resistant S. aureus
(MRSA) strains has become an important cause of health care-associated MRSA (HA-
MRSA), community-associated MRSA (CA-MRSA), and livestock-associated MRSA (LA-
MRSA) infections. The ability of S. aureus to colonize the nares and the skin of humans and
animals, and the detection of MRSA and MDR strains in meat-producing animals and retail
meat, have increased the probability of contamination of food in different steps of the food
production chain. Methicillin resistance is attributed to the penicillin-binding protein 2a
(PBP2a) encoded in the mecA gene, which is carried on the staphylococcal cassette
chromosome mec (SCCmec). This protein gives MRSA strains a reduced affinity for β-lactam
antibiotics. CA-MRSA strains are more likely to encode the Panton–Valentine leukocidin
(PVL) exotoxin, which is a virulence factor related to skin infections and tissue necrosis.
Therefore, since the impact of those S. aureus strains in human health it is important to
evaluate whether food could be a source of contamination that facilitate the spread of
infections.
The aim of this study was to stablish the prevalence and molecular types of S. aureus
and MRSA from meat-producing animals, retail raw meat, deli meat, and humans, determining
the phylogenetic relationship between the strains.
A two-step selective enrichment followed by a culture method were used to isolate S.
aureus from 167 nasal swabs from animals, 145 samples of retail raw meat, and 46 samples of
deli meat. Positive isolates were subjected to multiplex PCR (mPCR) in order to identify the
genes 16S rRNA (identification of S. aureus), mecA (detection of MRSA) and PVL-encoding
genes (virulence factor associated to CA-MRSA). Pulsed-field gel electrophoresis (PFGE) and
multilocus sequence typing (MLST) were used for molecular typing of S. aureus strains.
6
Antimicrobial susceptibility testing was carried out using the broth microdilution method. In
addition, S. aureus from nasal swabs obtained from 550 healthy subjects was isolated and
identified by culture method and biochemical testing. A total of 108 MRSA isolates recovered
from hospital patients were also examined by mPCR to confirm S. aureus and to detect S.
aureus harboring the mecA and PVL genes. A multiplex real-time PCR assay was developed
in order to decrease the time of detection in animal and meat samples and increase the
sensibility of detection of S. aureus and the mecA and PVL genes. This procedure was carried
out after the primary and the secondary enrichments that preceding the culture method. The
results were compared with those obtained by the culture method which includes the two-step
selective enrichment in order to isolate S. aureus. A total of 234 samples were examined by
both methods (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat).
The overall prevalence of S. aureus determined by the culture method with two-step
selective enrichment was 34.7% in animals, with the highest rate found in pigs (50.0%) and
sheep (40.6%) (P<0.05). In raw meat the prevalence was 47.6%, with the highest rate in
chicken (67.6%) and pork (49.3%) (P<0.05). In deli meat the prevalence was 13.0% (Figure
2). Five pork samples (7.0%) were positive for mecA gene, of which three were serotype (ST)
ST398 and two were ST5. All these strains exhibited penicillin resistance and four were MDR
strains. The PVL genes were not detected in any sample by mPCR. The most common
serotypes in sheep were ST398 and ST133, in pigs and pork meat both ST398 and ST9, and in
chicken ST5. A genetic similarity between S. aureus strains ST9 isolated from pigs and pork
meat was obtained. Most susceptible S. aureus strains to antimicrobial agents were ST5
isolated from chicken. The MDR isolates were found in pigs, pork meat, and sheep.
The prevalence of nasal carriage of S. aureus in healthy people was 7.6%. None of
these strains were mecA- nor PVL-positive. A total of 105 MRSA strains (97.2%) obtained
from hospital patients harbored the mecA gene and 11 (10.2%) PVL genes.
7
0
10
20
30
40
50
60
70
80P
reva
lenc
e (%
)
Sheep
Pig
Cow
Pork
Chicken
Beef
Pork deli meat
Turkey deli meat
Chicken deli meat
Figure 2. Prevalence of Staphylococcus aureus in animals, raw meat, and deli meat.
The application of real-time PCR assay on samples following primary and secondary
enrichments detected S. aureus in 111/234 and 120/234 samples, respectively. These detection
levels were higher than recovery obtained by the culture method, which was 95/234. Total
agreement with the culture method for detection of S. aureus using real-time PCR was 83.9-
97.8% (kappa=0.68-0.88 [from substantial to almost perfect agreement]), and 68.9-88.3%
(kappa=0.29-0.77 [from fair to substantial agreement]) for primary and secondary enrichments,
respectively. For detection of the mecA gen, total agreement was 91.1-98.7% and 86.7-98.7%
(kappa=0-0.49 [from no agreement beyond that expected by chance to moderate agreement])
for primary and secondary enrichment samples, respectively. The real-time PCR assay
detected mecA gene in some samples that were negative for S. aureus, but positive for
Staphylococcus spp.
The presence of MRSA, MDR S. aureus, and the serotype ST398 in the meat
production chain indicates that there is a risk of transmission to humans. The genetic similarity
between strains of porcine origin (animals and meat) suggests the possible contamination of
meat during slaughtering. S. aureus harboring mecA and PVL genes is present in patients
affected by invasive infections. The rate of nasal carriage of S. aureus in healthy humans
Animals Raw meat Deli meat
8
seems to be low, without the presence of mecA and PVL genes, which represents a low risk of
transmission among the community. The real-time PCR assay may be recommended as a rapid
method for the detection of S. aureus and the mecA gen, with further confirmation of MRSA
using the standard culture method.
The results of this study suggest that an effective surveillance should be implemented
in the meat production chain in order to monitor MRSA and MDR S. aureus, and to take
actions to decrease the spread of AR strains.
Keywords: Methicillin resistant Staphylococcus aureus (MRSA), multidrug resistant (MDR),
mecA, Panton-Valentine Leukocidin (PVL), retail raw meat, animals, deli meat, clinical
isolates, healthy humans.
9
I. INTRODUCCIÓN GENERAL
La emergencia y propagación de patógenos resistentes a múltiples agentes
antimicrobianos ha aumentado la preocupación en materia de salud a nivel mundial. La
emergencia de cepas bacterianas resistentes a antibióticos en animales se relaciona con la
utilización de antibióticos en la etapa productiva (de Neeling et al., 2007). Numerosos agentes
antimicrobianos se suministran en la dieta de los animales con fines preventivos y de
promoción del crecimiento, lo cual expone a un gran número de animales a concentraciones
sub-terapeúticas de antibióticos en forma frecuente (DuPont y Steele, 1987; Franco et al.,
1990), aumentando la probabilidad de generar bacterias resistentes a antibióticos. Esto puede
tener como consecuencia que los genes de resistencia sean traspasados a bacterias presentes en
humanos, lo que representa un riesgo latente para la pérdida de eficacia de los antibióticos
utilizados en salud humana (Smith et al., 2002).
Existen evidencias sobre la contaminación de la carne con bacterias resistentes a
múltiples agentes antimicrobianos, como Salmonella, Campylobacter, Enterococcus y
Escherichia coli (FDA, 2010). En animales y en carne se encontraron cepas de Staphylococcus
aureus resistente a meticilina (SARM) y S. aureus multirresistente (MR) (Waters et al., 2011;
de Neeling et al., 2007). Sin embargo, no se tiene suficiente información sobre la prevalencia
de SARM y S. aureus MR en alimentos de origen animal.
En humanos, S. aureus puede ocasionar una amplia gama de enfermedades como
intoxicación alimentaria, neumonía, infecciones de heridas e infecciones nosocomiales
(hospitalarias) (Tiemersma et al., 2004; Kennedy et al., 2008). Esta bacteria es un patógeno
oportunista que se propaga a través del contacto directo de animales o personas con heridas
infectadas o con otros procesos infecciosos. La transmisión también puede ocurrir desde
personas o animales colonizados por esta bacteria que son portadores asintomáticos (CFSPH,
2011).
Staphylococcus aureus y SARM pueden colonizar las fosas nasales y la piel de los
animales (de Neeling et al., 2007; Moon et al., 2007; Lewis et al., 2008; van Belkum et al.,
2008; Guardabassi et al., 2009; Persoons et al., 2009), lo cual aumenta el riesgo de
contaminación de las canales durante la faena de animales de abasto (de Boer et al., 2009).
10
En Estados Unidos se estima que la prevalencia de colonización nasal con S. aureus en
humanos es de aproximadamente del 29% y con SARM de. 1,5% (Gorwitz et al., 2008). Por lo
tanto, las personas también constituyen una fuente de contaminación durante la manipulación
de los alimentos. De esta forma, los alimentos contaminados por esta vía se transforman en
vehículos para diseminar este patógeno, siendo los de mayor riesgo aquellos sometidos a una
cocción inadecuada (CFSPH, 2011) y aquellos que no requieren cocción para su consumo.
En investigaciones anteriores se determinó la prevalencia y se tipificaron molecularmente
diferentes cepas de S. aureus y SARM en muestras de animales y carne (Waters et al., 2011;
de Neeling et al., 2007) y, en otras investigaciones en muestras de humanos (Tiemersma et al.,
2004; Kennedy et al., 2008), siendo difícil establecer la relación o similitud genética entre
ellas. Este estudio pretende establecer la prevalencia de S. aureus, cepas sensibles y resistentes
a la meticilina, en animales, carne, productos cárnicos y humanos y determinar la relación
filogenética de las cepas aisladas.
1. Características de Staphylococcus aureus
Inicialmente, el género Staphylococcus fue clasificado dentro de la familia
Micrococaceae. Sin embargo, estudios recientes de homología genética demostraron que los
géneros Staphylococcus y Micrococcus tienen poca relación. Por esta razón, Staphylococcus
se incluyó en la familia Staphylococcaceae, dentro del orden Bacillales (Euzéby, 1997). El
nombre del género -Staphylococcus- proviene del griego staphylé que significa racimo de uvas,
ya que presentan forma esférica (cocos), con un diámetro de 0,5 a 1,5 µm y se agrupan de
forma irregular. Éstas son bacterias gram positivas, inmóviles, no forman esporas y,
generalmente, no poseen cápsula y son anaerobias facultativas. Se diferencian de los géneros
Streptococcus y Enterococcus por producir la enzima catalasa, la cual hidroliza el peróxido de
hidrógeno (H2O2) en oxígeno (O2) y agua (H2O). Presentan metabolismo oxidativo y
fermentativo de la glucosa, a diferencia del género Micrococcus, el cual no la fermenta (de
Cueto y Pascual, 2009).
En un medio de cultivo no selectivo S. aureus presenta colonias lisas, solelevadas,
brillantes, de consistencia cremosa, de color amarillo o dorado, debido a la producción de un
pigmento carotenoide. Esta especie es resistente al calor y a la desecación y tiene la capacidad
11
de crecer en medios de alta salinidad (7,5% de NaCl). En medios de cultivo que contienen
sangre, la mayoría de las cepas produce lisis de los glóbulos rojos por acción de la β-
hemolisina (β-hemólisis), lo cual se manifiesta con un halo claro alrededor de las colonias.
Una de las características que diferencia a S. aureus de otras especies, salvo excepciones, es la
producción de la enzima coagulasa, que transforma el fibrinógeno en fibrina coagulando el
plasma sanguíneo (Lowy, 1998; de Cueto y Pascual, 2009). Adicionalmente, S. aureus
produce una ADNasa termoestable que rompe los enlaces fosfodiéster del ADN. Esta
propiedad sirve como método de identificación, dado que al someter a S. aureus a altas
temperaturas y luego incubar con ADN, se observará la degración de ADN (de Cueto y
Pascual, 2009).
Staphylococcus aureus presenta diferentes factores de virulencia. Posee componentes
microbianos de superficie que reconocen moléculas adhesivas de la matriz (MSCRAMM, del
inglés microbial surface components recognizing adhesive matrix molecules), las cuales
median la adherencia a los tejidos, tales como el factor de agregación (clumping factor),
proteínas de unión al fibrinógeno, fibronectina y sialoproteína ósea (Lowy, 1998; de Cueto y
Pascual, 2009). Además, esta bacteria produce un polisacárido de adherencia intracelular que
permite su persistencia a través de la formación de una biocapa bacteriana que le confiere
protección (de Cueto y Pascual, 2009). Para evadir los mecanismos de defensa del huésped, S.
aureus presenta la proteína A, proteína de adherencia extracelular y citotoxinas (Leucocidina
de Panton-Valentine [PVL], α-toxina). Adicionalmente, sintetiza lipasas, hialuronidasas y
proteasas, las que destruyen los tejidos, facilitando la propagación de la infección. Otros
factores de virulencia son los asociados a las intoxicaciones alimentarias y shock tóxicos:
enterotoxinas, toxina del síndrome del shock tóxico 1, toxinas exfoliativas A y B y α-toxina
(Lowy, 1998; de Cueto y Pascual, 2009). Todos estos factores de virulencia presentes en S.
aureus promueven la colonización e invasión, ocasionando un daño severo al hospedero.
2. Mecanismos de resistencia a meticilina
Existen muchos agentes con actividad antiestafilocócica, sin embargo, S. aureus ha
desarrollado mecanismos para neutralizarlos, encontrándose cepas resistentes a múltiples
agentes antimicrobianos (McDougal et al., 2003; Aydin et al., 2011; Waters et al., 2011).
12
En Europa, a principios de la década de los 60s, poco después de la introducción de la
meticilina (penicilina semisintética resistente a penicilinasa) surgió S. aureus resistente a
meticilina (SARM), asociado a infecciones originadas en instalaciones hospitalarias. A fines
de la década de los 90s, emergieron en todo el mundo cepas de SARM asociados a infecciones
originadas en la comunidad (Lowy, 1998, 2003; Deurenberg y Stobberingh, 2008). En
consecuencia, la propagación global de SARM ha generado preocupación mundial (Voss y
Doebbling, 1995), debido a la aparición creciente de infecciones nosocomiales (Tiemersma et
al., 2004), comunitarias (Kennedy et al., 2008) y en animales (Golding et al., 2010).
La meticilina es un antibiótico β-lactámico, al igual que la penicilina G, oxacilina,
ampicilina, amoxicilina y cefalosporinas, entre otros. Estos antibióticos inhiben la síntesis de
la pared celular de bacterias gram positiva, mediante la inhibición de la última etapa de la
síntesis del peptidoglicano, correspondiente a la unión del ácido N-acetilmurámico a la pared
celular, lo que se conoce también como transpeptidación. La transpeptidación está catalizada
por transpeptidasas y carboxipeptidasas, llamadas también proteínas de unión a penicilina
(PBPs) por su capacidad para fijar penicilina en su sitio activo. El anillo β-lactámico se une de
forma covalente a una serina presente en el sitio activo de las PBPs, inactivando la
transpeptidación. Así, la pared celular queda debilitada y puede romperse por la presión
osmótica intracelular. Existen PBPs que inhiben enzimas autolíticas de la pared bacteriana
(autolisinas), por lo cual al unirse la penicilina al sitio de fijación de las PBPs, no se inhiben
las autolisinas produciendo la lisis celular (Marín y Gudiol, 2003; Romero, 2007).
Uno de los mecanismos de resistencia a antibióticos β-lactámicos corresponde a la acción
de la enzima β-lactamasa, la cual confiere resistencia a la penicilina, debido a que hidroliza el
anillo β-lactámico, inactivando al antibiótico. Esta enzima está codificada en el gen blaZ, que
se encuentra en un transposón ubicado en un plásmido junto con otros genes asociados a
resistencia antimicrobiana (Lowy, 2003).
La resistencia a la meticilina presente en SARM confiere resistencia a todas las
penicilinas resistentes a penicilinasa y cefalosporinas (Lowy, 1998). Esto se atribuye a la
proteína de unión a la penicilina 2a (PBP2a, penicillin-binding protein 2a), que posee baja
afinidad por los antibióticos β-lactámicos (Hartman y Tomasz, 1981; Lim y Strynadka, 2002).
La proteína PBP2a se diferencia de otras PBPs en que su sitio activo no permite la fijación de
13
antibióticos β-lactámicos, posibilitando el desarrollo de la reacción de transpeptidación. De
esta manera, al formarse la pared celular, se favorece la supervivencia de los estafilococos
expuestos a altas concentraciones de estos agentes antimicrobianos (Lim y Strynadka, 2002).
La proteína PBP2a está codificada por el gen mecA, el cual se encuentra en un elemento
genético móvil denominado cassette cromosómico estafilocócico mec (SCCmec) (Hartman y
Tomasz, 1981). La expresión del gen mecA es regulada por dos proteínas: MecI, represor
codificado por el gen mecI; y MecR1, proteína transductora de señal codificada por el gen
mecR1. En ausencia de antibióticos β-lactámicos, la proteína MecI se une al operador,
reprimiendo la transcripción del gen mecA. La fijación de antibióticos β-lactámicos a la
proteína MecR1, provoca la liberación de un fragmento con actividad proteasa de esta proteína,
el cual divide al represor MecI en fragmentos inactivos, permitiendo la transcripción del gen
mecA y la posterior síntesis de PBP2a (Lowy, 2003).
3. Tipificación molecular de Staphylococcus aureus y SARM
La tipificación de las cepas de S. aureus no está completamente estandarizada y a través
de los años se han utilizado una gran variedad de métodos (Tenover et al., 1994). Dentro de las
técnicas moleculares que se utilizan actualmente para tipificar las cepas de SARM está: la
electroforesis en gel de campo pulsado (PFGE), basado en la macro-restricción del ADN
genómico; la tipificación de secuencias multilocus (MLST), basada en el perfil alélico de siete
genes conservados; y la tipificación de spa, basado la secuenciación de la región X del gen
que codifica la proteína A presente en S. aureus (McDougal et al., 2003). Por esta razón, una
misma cepa puede recibir diferentes nombres (CFSPH, 2011). Los Centros para el Control y la
Prevención de Enfermedades (CDC, del inglés Centers for Disease Control and Prevention)
establecieron un sistema de nomenclatura para S. aureus y SARM comunes en Estados Unidos,
basado en los patrones de macro-restricción obtenidos a través de PFGE, identificando ocho
tipos, desde USA100 hasta USA800 (McDougal et al., 2003). La nomenclatura utilizada para
MLST corresponde a serotipos (ST) seguido de un número (ej. ST398), mientras que para
tipificación de spa se utiliza "t" seguido de un número (ej. t011) (Cuny et al., 2010).
Generalmente, PFGE y MLST clasifican las cepas en clusters similares (Catry et al., 2010), los
cuales pueden contener diferentes tipos spa. Una de las desventajas de la tipificación de spa es
que líneas clonales no relacionadas pueden tener tipos spa similares (Van den Broek IV et al.,
14
2009; Golding et al., 2008). Esto se debe a que la tipificación de spa se centra en una pequeña
fracción del genoma y, una recombinación, podría resultar en discrepancias con los clusters
obtenidos a través de MLST y PFGE (Golding et al., 2008). Se ha demostrado que el método
PFGE tiene mayor poder de discriminación que MLST y la tipificación de spa. Sin embargo,
estas técnicas pueden utilizarse para determinar cambios mayores que pueden ocurrir en las
líneas clonales a través del tiempo (McDougal et al., 2003). Para lograr una mayor precisión
en la tipificación de las cepas se recomienda la combinación de dos métodos (Tenover et al.,
1994).
Con la utilización de PFGE se determinó que las cepas de SARM causantes de
infecciones adquiridas en la comunidad (USA300 y USA400) son diferentes a las cepas
nosocomiales (USA100 y USA200) (McDougal et al., 2003). Vandenesch et al. (2003)
determinaron que las cepas de SARM comunitario provenientes de tres continentes comparten
dos genes, el cassette cromosómico SCCmec tipo IV y el locus PVL, que contiene los genes
productores de la toxina PVL. Los genes PVL están localizados en un bacteriófago que infecta
a S. aureus, mientras que la distribución de otros genes de toxinas es específica de las cepas de
cada continente. La mayoría de las cepas de SARM comunitario presentan el locus PVL (Baba
et al., 2002; Dufour et al., 2002), factor de virulencia asociado a infecciones de piel, neumonía
y necrosis de tejidos (Ebert et al., 2009).
A través del método MLST se ha podido determinar la presencia de algunos tipos de
secuencias asociadas a SARM hospitalario, como ST5, ST8, ST22, ST36, ST45, entre otros
(Deurenberg et al., 2007); mientras que ST30 y ST80 han sido asociados a SARM comunitario
(Stenhem et al., 2010) y ST398 a SARM en animales, específicamente en cerdos (Lewis et al.,
2008; van Belkum et al., 2008; Krziwanek et al., 2009). El serotipo ST398, inicialmente
asociado a cerdos, ha sido encontrado en personas, la mayoría productores de cerdos (van
Belkum et al., 2008; Krziwanek et al., 2009; Pan et al., 2009; Golding et al., 2010). Además,
recientemente se asociaron infecciones a SARM ST398 en humanos en contacto con vacas
lecheras con mastitis sub-clínica (Soavi et al., 2010). No obstante, en Suecia, se reportaron dos
casos de ST398 t038 en pacientes que no tuvieron contacto con animales (Welinder-Olsson et
al., 2008). Esto sugiere la propagación de estas cepas en el ambiente, facilitando la
colonización de personas que no están involucradas con la producción animal, generando una
creciente preocupación en salud pública (Gibbs et al., 2006). Staphylococcus aureus resistente
15
a meticilina ST398 no es tipificable mediante PFGE, debido a que su ADN no puede ser
digerido por la enzima de restricción SmaI, ya que posee una enzima que metila la secuencia
de reconocimiento de SmaI (Bens et al., 2006). Análisis comparativos de la huella genética de
cepas de SARM ST398 demostraron que existe diversidad molecular y geográfica (Golding et
al., 2010). Además, existe evidencia que demuestra la aparición de cepas de SARM diferentes
a ST398, como ST9 t899, asociado también a la producción de cerdos (Guardabassi et al.,
2009). Por lo tanto, la emergencia de nuevas cepas de SARM en cerdos resalta la importancia
de establecer estrategias de vigilancia permanente, determinando el riesgo de transmisión a las
personas relacionadas con producción porcina.
4. Prevalencia de Staphylococcus aureus y SARM
El método para aislar S. aureus y SARM no está estandarizado. Por lo tanto, la utilización
de diferentes métodos puede afectar la sensibilidad de la detección y, por consiguiente, los
resultados de prevalencia. Algunos análisis han incluido solamente cultivo en placa utilizando
agar manitol sal (MSA) con 2 µg/mL de oxacilina (Weese et al., 2006) o agar Baird Parker
(BP) (Aydin et al., 2011). Otros estudios han incorporado etapas de enriquecimiento previo al
cultivo en placa. Wertheim et al. (2001) desarrollaron un caldo selectivo con rojo fenol,
manitol, aztreonam y ceftizoxima (PHMB+), lo cual aumentó al doble la sensibilidad de
detección de SARM. Broens et al. (2011) utilizaron un protocolo de dos etapas de
enriquecimiento, una con caldo Mueller-Hinton con 6,5% de NaCl (MHB+6,5%NaCl) y la
otra con PHMB+, seguido de cultivo en agar cromogénico selectivo para SARM. Otros autores
han utilizado enriquecimiento en caldo con 7,5% de NaCl, 1% de manitol y 2,5% de extracto
de levadura seguido de un medio cromogénico (Zhang et al., 2011); caldo tripticasa soya
suplementado con 10% de NaCl y 1% de piruvato de sodio seguido de cultivo en agar BP (Pu
et al., 2009; Pu et al., 2011); PHMB+ seguido de cultivo en agar con sangre ovina y dos agares
selectivos (Tenhagen et al., 2009). Estos distintos métodos sugieren que para aumentar la
sensibilidad de detección de S. aureus y SARM se debería incluir una etapa de
enriquecimiento seguida de un cultivo selectivo.
La mayoría de los animales pueden ser colonizados con S. aureus, encontrándose en las
fosas nasales y la piel (de Neeling et al., 2007; Moon et al., 2007; Lewis et al., 2008; van
Belkum et al., 2008; Guardabassi et al., 2009; Persoons et al., 2009). Por esta razón, durante la
16
faena existe el riesgo de contaminación de las canales y la carne con S. aureus y SARM (de
Boer et al., 2009). Recientemente, se aislaron cepas de SARM en cerdos, vacunos y pollos (de
Neeling et al., 2007; Moon et al., 2007; Lewis et al., 2008; van Belkum et al., 2008;
Guardabassi et al., 2009; Persoons et al., 2009). En Holanda de Neeling et al. (2007)
encontraron una alta prevalencia de SARM ST398 (39%) en cerdos y una alta tasa de
resistencia a diferentes antibióticos (tetraciclina, eritromicina, clindamicina, kanamicina,
gentamicina, tobramicina) sugiriendo, además, la existencia de contaminación entre los
animales en los corrales de las plantas faenadoras. En un estudio realizado en Hong Kong se
determinó una prevalencia de SARM en cerdos menor (16%) a la reportada en el estudio
anterior (Guardabassi et al., 2009), lo que podría deberse al tamaño menor de la muestra
analizada y al método utilizado en la detección. Dado que los cerdos constituyen una posible
fuente de infección de SARM se requiere estudiar la epidemiología de esta zoonosis
emergente, determinando la frecuencia de transmisión de SARM desde animales a humanos, y
la transmisión persona-persona (Lewis et al., 2008).
En los últimos años se han aislado cepas de SARM en carne de cerdo, pollo, vacuno,
pavo y cordero. Hanson et al. (2011) analizaron diferentes tipos de carne de supermercados del
Estado de Iowa, Estados Unidos, encontrando sólo 2 muestras de carne de cerdo contaminadas
con cepas de SARM, con una prevalencia total de alrededor de 1%. En Estados Unidos, un
estudio realizado en el Estado de Louisiana reveló contaminación con S. aureus en 45,6% de
las muestras de carne de cerdo y en 20% de las muestras de carne de vacuno, de los cuales el
5,6% y el 3,3% correspondió a cepas de SARM en carne de cerdo y vacuno, respectivamente
(Pu et al., 2009). de Boer et al. (2009) encontraron una mayor porporción de carne
comercializada en supermercados de Holanda contaminada con SARM, principalmente ST398,
de alrededor de un 35% en carne de pavo, 16% en carne de pollo, 11% en carne de cerdo, 10%
en carne de vacuno y 6% en carne de cordero. Estos últimos valores de prevalencia de SARM
en carne sugieren que el método utilizado en aquél estudio (dos etapas de enriquecimiento
selectivo + cultivo selectivo) permitió una mayor sensibilidad de detección de SARM.
Finalmente, la detección de SARM en carne ha aumentado la preocupación en materia de
inocuidad alimentaria con respecto a la cadena de producción de carne y ha generado la
urgencia de contar con sistemas de vigilancia y coordinación entre diferentes laboratorios de
análisis.
17
En Estados Unidos, una de cada seis personas contrae una enfermedad transmitida por
alimentos cada año, con un total de 48 millones de personas afectadas. De los agentes
causantes, S. aureus es uno de los cinco principales, con un total de 240 mil casos,
correspondientes a una enfermedad gastrointestinal causada por el consumo de toxinas
producidas por S. aureus (CDC, 2012). Por otro lado, las cepas de SARM están asociadas
principalmente a infecciones de la piel en la comunidad, y los casos más graves se asocian a
pacientes hospitalizados, afectados por infecciones sanguíneas, quirúrgicas, o neumonía (CDC,
2011). Aproximadamente el 29% de la población en Estados Unidos es portadora de S. aureus
y el 1,5% de SARM en las fosas nasales (Gorwitz et al., 2008). Del total de 478.000 casos de
infecciones por S. aureus que resultaron en hospitalizaciones en el año 2005,
aproximadamente el 50% se asoció a SARM, y del total de 11.406 muertes asociadas a S.
aureus, 6.639 correspondieron a infecciones por SARM (Klein et al., 2007).
En el Estado de Dakota del Norte se cuenta con información epidemiológica acerca de las
infecciones causadas por SARM en humanos. Sin embargo, no existe información acerca de la
prevalencia de cepas de SARM en animales o carne. En el año 2011, se registraron 13 casos
de infecciones asociadas a SARM por cada 100.000 habitantes. Estas infecciones aumentaron
desde el año 2001 hasta el año 2006, manteniéndose actualmente sin variación (North Dakota
Department of Healt, Disease Control, 2011).
Las infecciones causadas por microorganismos resistentes a antibióticos tienen un costo
económico considerablemente alto. En promedio, el costo de hospitalización por infecciones
de SARM es de aproximadamente US$ 14.000, comparado con US$ 7.600 de hospitalización
por infecciones no-SARM. Por lo tanto, el costo total de las hospitalizaciones por infecciones
de SARM en Estados Unidos asciende a más de 3 billones de dólares al año. (Elixhauser y
Steiner, 2007). Lo anterior no incluye las pérdidas que se generan por ausentismo laboral,
incapacidad y mortalidad.
La prevención y control de infecciones de SARM en Estados Unidos está dirigida por el
CDC, que proporciona información específica para cada grupo de la población y estrategias
para el tratamiento de infecciones de SARM en la comunidad. La disminución de infecciones
de SARM en hospitales y en la comunidad es de alta prioridad para CDC. Es así como se
establecieron proyectos de vigilancia a corto y largo plazo con la colaboración de los
18
departamentos de salud, hospitales, centros médicos, entre otros (CDC, 2011). Sin embargo, se
necesitan más estudios para poder caracterizar genéticamente las cepas de SARM y determinar
las rutas de transmisión en animales y humanos, lo que facilitará el desarrollo de acciones
preventivas para disminuir su propagación.
5. Hipótesis y objetivos
Hipótesis
Staphylococcus aureus, cepas sensibles y resistentes a la meticilina, están presentes en
animales, carne, y humanos, existiendo relación filogenética entre las cepas.
Objetivo general
Establecer la prevalencia y los tipos moleculares de S. aureus y SARM en animales de abasto,
carne cruda, productos cárnicos y humanos, determinando la relación filogenética entre las
cepas.
Objetivos específicos
- Determinar la presencia de cepas de S. aureus en animales, carne, productos cárnicos y
humanos.
- Determinar la prevalencia de S. aureus y SARM en animales, carne, productos cárnicos y
humanos.
- Determinar características moleculares de las cepas de S. aureus y SARM a través de las
técnicas de PCR múltiple, electroforesis en gel de campo pulsado y análisis de secuencias
multilocus.
- Determinar el perfil de resistencia antimicrobiana de las cepas de S. aureus y SARM.
- Comparar la técnica de PCR en tiempo real con el método de cultivo y PCR convencional,
utilizando dos etapas de enriquecimiento selectivo, para la detección de S. aureus y SARM
en muestras de animales, carne y productos cárnicos.
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6. Referencias
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Baba, T., F. Takeuchi, M. Kuroda, H. Yuzawa, K. Aoki, A. Oguchi, Y. Nagai, N. Iwama, K.
Asano, T. Naimi, H. Kuroda, L. Cui, K. Yamamoto, K. Hiramatsu. 2002. Genome and
virulence determinants of high virulence community-acquired MRSA. Lancet. 359, 1819-
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Bens, C.C.P.M., A. Voss, C.H.W. Klaassen. 2006. Presence of a novel DNA methylation
enzyme in methicillin-resistant Staphylococcus aureus Isolates associated with pig farming
leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis. J. Clin.
Microbiol. 44, 1875-1876.
Broens, E.M., E.A. Graat, B. Engel, R.A. van Oosterom, A.W. van de Giessen, P.J. van der
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27
II. DESARROLLO
1. Molecular typing of Staphylococcus aureus and methicillin-resistant S.
aureus (MRSA) isolated from animals and retail meat in North Dakota,
United States
Esra Buyukcangaz1,*, Valeria Velasco2,3,*, Julie S. Sherwood2, Ryan M. Stepan2, Ryan J.
Koslofsky2 and Catherine M. Logue2,4
Publicado en Foodborne Pathogens and Diseases, 2013, Volumen 10, Número 7, 608-617.
Abstract
The objective of this study was to determine the prevalence and molecular typing of
methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus
(MRSA) in food-producing animals and retail meat in Fargo, North Dakota. A two-step
enrichment followed by culture methods were used to isolate S. aureus from 167 nasal swabs
from animals, 145 samples of retail raw meat, and 46 samples of deli meat. Positive isolates
were subjected to multiplex polymerase chain reaction in order to identify the genes 16S
rRNA, mecA, and Panton-Valentine Leukocidin. Pulsed-field gel electrophoresis and
multilocus sequence typing were used for molecular typing of S. aureus strains. Antimicrobial
susceptibility testing was carried out using the broth microdilution method. The overall
prevalence of S. aureus was 37.2% (n=133), with 34.7% (n=58) of the animals positive for the
organism, and the highest prevalence observed in pigs (50.0%) and sheep (40.6%) (p<0.05);
47.6% (n=69) of raw meat samples were positive, with the highest prevalence in chicken
(67.6%) and pork (49.3%) (p<0.05); and 13.0% (n=6) of deli meat was positive. Five pork
samples (7.0%) were positive for MRSA, of which three were ST398 and two were ST5. All
exhibited penicillin resistance and four were multidrug resistant (MDR). The Panton-
Valentine Leukocidin gene was not detected in any sample by multiplex polymerase chain
1Department of Microbiology, College of Veterinary Medicine, Uludag University, Bursa, Turkey. 2Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, North Dakota. 3Department of Animal Sciences, University of Concepción, Chillán, Chile. 4Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State
University, Ames, Iowa. *These two authors contributed equally to this work.
28
reaction. The most common clones in sheep were ST398 and ST133, in pigs and pork meat
both ST398 and ST9, and in chicken ST5. Most susceptible S. aureus strains were ST5
isolated from chicken. The MDR isolates were found in pigs, pork, and sheep. The presence of
MRSA, MDR, and the subtype ST398 in the meat production chain and the genetic similarity
between strains of porcine origin (meat and animals) suggest the possible contamination of
meat during slaughtering and its potential transmission to humans.
Address correspondence to: Catherine M. Logue, PhD Department of Veterinary Microbiology and Preventive Medicine 1802 University Boulevard, VMRI #2 College of Veterinary Medicine Iowa State University Ames, IA 50011
E-mail: [email protected]
1.1 Introduction
Outbreaks caused by antimicrobial-resistant (AR) bacteria is an established problem
worldwide (DeWaal et al., 2011). One of these AR pathogens is methicillin-resistant
Staphylococcus aureus (MRSA), which causes health care-associated MRSA (Tiemersma et
al., 2004), community-associated (CA-MRSA) (Kennedy et al., 2008), and livestock-
associated MRSA infections (Golding et al., 2010).
Most animals can become colonized with S. aureus (de Neeling et al., 2007; Moon et al.,
2007; Lewis et al., 2008; van Belkum et al., 2008; Guardabassi et al., 2009; Persoons et al.,
2009), and contamination of carcasses may occur during slaughtering (de Boer et al., 2009).
Recently, MRSA strains have been isolated from several food-producing animals (de Neeling
et al., 2007; Moon et al., 2007; Lewis et al., 2008; van Belkum et al., 2008; Guardabassi et al.,
2009; Persoons et al., 2009); and from retail meat worldwide (de Boer et al., 2009; Pu et al.,
2009; Lim et al., 2010; Weese et al., 2010; Bhargava et al., 2011; Hanson et al., 2011),
representing a potential risk for its transmission to humans.
Methicillin resistance is attributed to the altered penicillin binding protein (PBP2a), encoded
in the mecA gene, which has a reduced affinity for β-lactam antibiotics (Hartman and Tomasz,
29
1981; Van De Griend et al., 2009). CA-MRSA strains are more likely to encode a virulence
factor called Panton-Valentine leukocidin (PVL) toxin (Baba et al., 2002; Dufour et al., 2002),
associated with skin infections and tissue necrosis (Ebert et al., 2009). Therefore, the PVL
toxin has been identified as a genetic marker for CA-MRSA strains (Vandenesch et al., 2003).
Different molecular techniques have been used for typing MRSA strains, such as pulsed-field
gel electrophoresis (PFGE) based on macrorestriction patterns of genomic DNA; multilocus
sequence typing (MLST) that determines the allelic profile of seven housekeeping genes; and
spa typing based on the sequencing of the polymorphic X region of the protein A gene. It has
been demonstrated that the discriminatory power of PFGE is greater than MLST and spa
typing (McDougal et al., 2003; Malachowa et al., 2005). Tenover et al. suggest that a
combination of two methods may provide more precision in epidemiological studies (Tenover
et al., 1994).
It has been demonstrated that MRSA strains causing CA-MRSA infections (USA300 and
USA400) are different from those causing health care-associated MRSA infections (USA100
and USA200) (McDougal et al., 2003). The sequence type ST398 has been associated with
livestock-associated MRSA (Lewis et al., 2008; van Belkum et al., 2008; Welinder-Olsson et
al., 2008; Krziwanek et al., 2009), however, the presence of ST398 and the emergence of
infections in humans with livestock exposure, mostly pig farmers, has increased the public
health concern (van Belkum et al., 2008; Krziwanek et al., 2009; Pan et al., 2009; Golding et
al., 2010).
The aim of this study was to determine the prevalence, molecular typing, and genetic
similarity of S. aureus and MRSA isolated from animals and retail meat in Fargo, ND.
1.2 Materials and Methods
1.2.1 Samples
A total of 167 nasal swabs (sheep, n = 64; pigs, n = 60; cows, n = 43) were collected from
food-producing animals immediately after stunning at the Meat Lab (Department of Animal
Sciences). Of these samples a total of 57 (sheep, n = 14; pigs, n = 18; cows, n = 25) were
30
obtained from sick animals at the ND Veterinary Diagnostic Lab (North Dakota State
University, Fargo, ND).
Moreover, 145 raw meat (pork, n = 71; chicken, n = 37; beef, n = 37) and 46 deli meat (ham, n
= 21; turkey, n = 16; chicken, n = 9) samples were randomly purchased from four supermarket
chains in Fargo, ND.
Samples were collected between May 2010 and April 2011, immediately stored at 4°C, and
processed within 6 h of collection.
1.2.2 Isolation of S. aureus and MRSA
The isolation was carried out by enrichment (de Boer et al., 2009) followed by plating steps on
selective agar. Briefly, for the primary enrichment, 25 g of meat and 225 mL of Mueller-
Hinton broth (Becton, Dickinson and Company [BD], Sparks, MD) with 6.5% sodium
chloride (VWR International, West Chester, PA) (MHB+6.5% NaCl) were placed in a sterile
stomacher bag and homogenized using a stomacher400 circulator (Seaward, England) at 230
rpm for 90 s. The suspension was incubated for 18-20 h at 37°C. One milliliter of primary
enrichment was inoculated into 9 mL of phenol red mannitol broth (BD) containing
ceftizoxime (5 µg/mL, US Pharmacopeia, Rockville, MD) and aztreonam (75 µg/mL, Sigma
Chemical Co., St. Louis, MO) (PHMB+) (Wertheim et al., 2001), followed by incubation for
18-20 h at 37°C.
Nasal swabs were placed directly in 9 mL MHB+6.5% NaCl and incubated for 18-20 h at
37°C. Then, the procedure described above was carried out.
A loopful of secondary enrichment was struck directly to Baird-Parker medium with egg yolk
tellurite supplement (BP) (according to manufacturer’s recommendations) (BD) and incubated
for 48 h at 37°C. Two presumptive S. aureus colonies on BP (black colonies surrounded by 2-
to 5-mm clear zones) were transferred to Trypticase soy agar with 5% sheep blood (TSAII 5%
SB) (BD) and incubated for 18-20 h at 37°C. Presumptive S. aureus on TSAII 5% SB
(presence of β-hemolysis) was confirmed using Sensititre Gram Positive ID (GPID) plates
(Sensititre, TREK Diagnostic Systems Ltd., Cleveland, OH). Confirmed colonies were stored
frozen at -80°C in brain-heart infusion broth (BD) containing 20% glycerol until use.
31
1.2.3 Multiplex polymerase chain reaction (PCR)
All S. aureus strains were recovered from frozen stock to TSA plates and incubated at 37°C
for 18-24 h. DNA extraction was carried out by suspending one colony in 50 µL of
DNase/RNase-free distilled water, heating the suspension (99°C, 10 min) and then
centrifugation (30,000×g, 1 min) to remove cellular debris. The remaining DNA was
transferred to a new tube and stored frozen at - 20°C.
Multiplex PCR assay for detection of 16S rRNA, mecA and PVL genes included 2 µL of the
DNA template (described above) added to a 50 µL final reaction mixture: 1X Go Taq
Reaction Buffer (Promega, Madison, WI), 0.025 U/µL of Go Taq DNA polymerase
(Promega), 200 µM dNTP (Promega), and 1 µM of primers (16S rRNA, mecA, LukS/F-PV,
Table 1) (Integrated DNA Technologies, Inc., Coralville, IA) (McClure et al., 2006).
Multiplex PCR settings were carried out according to Makgotlho et al. (2009), using a
thermocycler (Eppendorf, Hamburg, Germany).
Ten microliters of the PCR amplicons were loaded into a 1.5% (wt/vol) agarose gel (Agarose
ITM, Amresco, Solon, OH) in 1X TAE buffer using EzVision One loading dye (Amresco),
and run at 100V in 1X TAE buffer for 1 h. A molecular weight marker 100-bp ladder
(Promega) and a positive control (ATCC 35591) were included on each gel. Bands were
visualized using an Alpha Innotech UV imager (FluorChemTM).
1.2.4 PFGE
The PulseNet protocol with slight modifications was used (McDougal et al., 2003). Briefly,
frozen isolates were struck in TSA plates and incubated at 37°C for 18-24 h. A single colony
was inoculated into a second TSA plate and incubated at 37°C for 18-24 h. Colonies were
transferred to 5-mL polystyrene round-bottom tubes containing 2 mL of cell suspension buffer
(100 mM Tris HCl [pH 8.0], Invitrogen; and 100mM EDTA [pH 8.0],Gibco), adjusting the
concentrations to an absorbance of 0.9-1.1 in a spectrophotometer (Smart SpecTM plus, Bio-
Rad Laboratories, USA) at 610 nm. After that, the preparation, lysis, and washes of plugs, and
32
then the SmaI enzyme restriction digestion were performed according to the PulseNet
protocol. Salmonella Branderup H9812 was used as a DNA marker (Ribot et al., 2006).
The electrophoresis was carried out in a Chef Mapper (Bio-Rad Laboratories) PFGE rig, with
an initial switch time of 5 s, a final switch time of 40 s, and a total running time of 17 h 45
min.
After staining the gels with ethidium bromide (1.5 µg/mL), they were visualized using a UVP
imager (UVP, Upland, CA). Macrorestriction patterns were compared using the BioNumerics
Fingerprinting software (Ver 6.5 Applied Math, Austin, TX). The similarity index was
calculated using the Dice coefficient, a band position tolerance of 1%, and an optimization of
0.5%. The unweighted-pair group method with arithmetic averages was used to construct a
dendrogram, and clusters were selected using a cutoff at 80%.
1.2.5 Multilocus sequence typing (MLST)
Briefly, S. aureus isolates were struck to TSA plates and incubated at 37°C for 18-24 h.
Colonies were picked to 40 µL of single cell lysing buffer (50 µg/mL of Proteinase K,
Amresco; in TE buffer [pH= 8]), and then lysed by heating to 80°C for 10 min followed by
55°C for 10 min in a thermocycler. The final suspension was diluted 1:2 in sterile water,
centrifuged to remove cellular debris, and transferred to a sterile tube (Marmur, 1961).
The housekeeping genes: arcC, aroE, glpF, gmk, pta, tpi, and yqiL, were amplified (Table 1)
(Enright et al., 2000). All PCR reactions were carried out in 50-µL volumes: 1 µL of DNA
template, Taq DNA polymerase (Promega) (1.25 U), 1X PCR buffer (Promega), primers (0.1
µM) (Integrated DNA Technologies, Inc.), and dNTPs (200 µM) (Promega). PCR settings
were adjusted according to Enright et al. (2000) using a thermocycler (Eppendorf). Ten
microliters of the PCR products were loaded into 1% agarose gels in 1X TAE with EzVision
One loading dye, and run at 100V in 1X TAE for 1 h. Images were captured using an Alpha
Innotech imager.
After PCR, each amplicon was purified of amplification primer using the QIAquick PCR
Purification Kit (Qiagen, Valencia, CA) as per manufacturer’s instructions. Purified DNA was
sequenced at Iowa State University’s DNA Facility (Ames, IA) using an Applied Biosystems
33
3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). Sequence data were imported
into DNAStar (Lasergene, Madison, WI), trimmed, and aligned to the control sequences (from
the MLST site) and interrogated against the MLST database (http://saureus.mlst.net/).
Sequence types were added to the strain information for analysis in BioNumerics.
1.2.6 Antimicrobial susceptibility testing
Minimum inhibitory concentrations (MICs) and the AR profiles of S. aureus isolates were
determined using the broth microdilution method (CMV3AGPF, Sensititre, Trek
Diagnostics), according to the manufacturer’s and the Clinical Laboratory Standards Institute
guidelines (CLSI, 2009).
A total of 16 antimicrobials belonging to 13 classes were tested. Resistance to at least three
classes of antibiotics was considered as multidrug resistance (MDR) (Aydin et al., 2011).
1.2.7 Statistical analysis
Fisher’s exact test was used to assess significance in prevalence of S. aureus and MRSA
between animal and meat types. A significance level of p<0.05 and two-sided p-values were
assessed using SAS software 9.2 (SAS Institute Inc., Cary, NC).
1.3 Results
Table 2 shows the prevalence of S. aureus in animals (34.7%, n=58), with a higher rate in
swine and sheep (p<0.05); in raw meat (47.6%, n=69), with a higher rate in chicken and pork
(p<0.05); and in deli meats (13.0%, n=6). MRSA was detected solely in meat (five pork
samples), representing a low prevalence (p<5%). The PVL gene was not detected in any
sample.
Most of the S. aureus isolates from animals were resistant to penicillin, tetracycline, and
lincomycin; and from raw meat to those antibiotics and erythromycin. All MRSA strains were
resistant to penicillin, and most of them showed resistance to erythromycin, tetracycline, and
lincomycin (Table 3).
34
A total of 47.7% (n=41) of the penicillin-resistant S. aureus strains exhibited MICs between
0.5 and 1 µg/mL. However, MRSA strains had higher MICs for penicillin (1- >16 µg/mL)
(Table 4).
The rate of MDR strains was 41.4% (n=55); in animals was 51.7% (n=30), and in meat was
36.2% (n=25). Among MRSA strains, only one was not MDR, and the rest showed MDR to
four classes of antimicrobials (Table 5).
Figure 1 shows a dendrogram displaying the macrorestriction patterns of S. aureus strains and
the sequence type (ST). The largest cluster (cluster 4) contained S. aureus of porcine origin
(animals and meat), all of which were ST9. S. aureus isolates included in the second largest
cluster (cluster 3) were obtained from poultry meat, and all but one was ST5. Two MRSA
isolates were clustered in cluster 5, all from pork and ST5. The rest of the MRSA isolates were
ST398 (not included in the dendrogram). A total of 34 S. aureus isolates (25.6%) were not
included in the dendrogram because they could not be restricted with SmaI or XmaI during
PFGE analysis and were ST398, isolated from animals (sheep and pigs) and from pork meat
(data not shown).
1.4 Discussion
Both methods used for the confirmation of S. aureus - Sensititre identification plates and
detection of the 16S rRNA gene by multiplex PCR- agreed with the results (Table 2). These
results confirmed that the isolation method of two enrichment steps preceding plating is an
appropriate method for recovering both S. aureus and MRSA from meat and animals. de Boer
et al. (2009) used the same two-step enrichment, reporting a higher detection rate of MRSA.
It is well known that animals are natural reservoirs of S. aureus; in this study, positive nasal
swabs were obtained from sheep, pigs, and cows. Other studies have detected a higher
prevalence of S. aureus in sheep (57%) and cattle (14%) (Mørk et al., 2012); however, the
prevalence in pigs has been reported to vary widely (6-57%) (Khalid et al., 2009; Lowe et al.,
2011). The recovery of S. aureus in meat in our study was higher than previous studies (39.2%
and 14.4%) (Pu et al., 2009; Aydin et al., 2011). The prevalence of S. aureus in ham was 19%,
which was considerably lower than the prevalence reported by Atanassova et al. (2001). There
35
is limited information about the prevalence of S. aureus and MRSA in processed retail meat
products, and this study provides some information as to the potential exposure of consumers
through consumption of deli meats that typically do not need heating prior to consumption.
In this study, MRSA was not detected in animals; however, a prevalence of MRSA in swine
ranging from 10% to 71% has been detected previously (Köck et al., 2009; Smith et al., 2009;
Tenhagen et al., 2009). The low rate of MRSA in pork raw meat (3.4%) determined in this
study agreed with the low prevalence reported by other authors (de Boer et al., 2009; Pu et al.,
2009).
Most of the S. aureus strains isolated from animals exhibited resistance to the same
antimicrobials reported by other authors (Nemati et al., 2008; Huber et al., 2010) (Table 3).
The AR bacteria in animals have increased over time due to the frequent use of antimicrobial
agents at the farm level (de Neeling et al., 2007; Nemati et al., 2008). Therefore, controlling
the use of antibiotics in farming could limit the risk of transmission of AR pathogens among
animals and potentially to humans (Huber et al., 2010).
Other authors have also determined a higher occurrence of resistance to penicillin,
tetracycline, and erythromycin in S. aureus strains isolated from retail meats and different
food samples (Aydin et al., 2011; Pu et al., 2011). Penicillin resistance has been reported to
spread rapidly among S. aureus strains being facilitated by plasmids and is the most frequently
reported resistance detected in foodborne S. aureus (Aydin et al., 2011).
AR S. aureus exhibited a MIC for erythromycin and lincomycin (>8 µg/mL) lower than the
MIC determined by Nemati et al. (2008). The MIC of tetracycline (>32 µg/mL) and penicillin
(0.5-1 µg/mL) concurred with the results reported by Nemati et al. (2008). Previously, other
authors have reported MDR in S. aureus from food samples at a lower rate compared with this
study (Aydin et al., 2011; Nam et al., 2011) (Table 4).
All S. aureus isolates examined in this study were susceptible to daptomycin, linezolid,
nitrofurantoin and vancomycin, concurring with the results reported by Pu et al. (2011).
The clustering of isolates obtained by PFGE agreed well with the MLST types (i.e., the
identical restriction patterns or patterns that differed at two to six bands had an identical ST)
36
(Fig. 1). Restriction patterns with the same numbers of bands represent the same strain;
patterns that differ up to three fragments represent strains that are closely related; and isolates
that differs at four to six bands may have the same genetic lineage (Tenover et al., 1995).
The major clones identified corresponded to ST9 and ST5. The emergence of ST9 in pigs was
first reported in 2008 by Guardabassi et al. (2009) in Hong Kong, disseminating later as
demonstrated in this study. The genetic relatedness between S. aureus strains ST9 from pigs
and pork meat may suggest the possible contamination of meat during slaughtering.
Previously, ST5 was associated with poultry (Hasman et al., 2010) and poultry meat (Waters
et al., 2011). In this study, the majority of strains isolated from chicken were ST5, which can
also suggest the contamination of meat during slaughtering. A high prevalence of ST398 was
found, which may indicate the potential risk for humans to acquire this emerging sequence
type that has potential for causing infection.
MRSA isolates had the same MLST allelic profile and indistinguishable PFGE patterns than
two methicillin-susceptible S. aureus (MSSA) strains, all obtained from pork. The close
genetic similarity of the MRSA and MSSA isolates may be due to the acquisition of the mecA
gene by horizontal transfer of SCCmec from MRSA strains to MSSA lineages (Enright et al.,
2000; Wielders et al., 2001; de Neeling et al., 2007; Guardabassi et al., 2009).
Most of the S. aureus isolates susceptible to all antimicrobial agents were obtained from
chicken, of which 76% were ST5. MDR isolates from pork were mainly ST398 (60%) (not
included in the dendrogram), and ST9 (30%). All MDR strains from sheep were ST398 (not
included in the dendrogram). The MDR can be due to the presence of other antibiotic
resistance genes, such as dfrK (resistance to trimethoprim) (Kadlec and Schwarz, 2009) and
cfr (MDR gene) (Kehrenberg et al., 2009).
1.5 Conclusion
The presence of MDR S. aureus, and subtype ST398 in animals and meat, and MRSA in retail
meat, and the genetic relationship between strains isolated from animals and meat, suggests
the likely contamination of meat during slaughtering.
37
Although the MRSA prevalence in raw meat is low, the prevalence of MDR S. aureus and
ST398 is higher; therefore, the risk of transmission through the meat production chain cannot
be ignored.
Acknowledgments
This study was supported by the Dean’s Office, College of Agriculture, Food Systems and
Natural Resources College, North Dakota State University (Valeria Velasco), the Dean’s
Office, College of Veterinary Medicine, Iowa State University, Ames, Iowa; and the
Commission of Scientific Research Projects, Uludag University (Project YDP (V)-2009/4).
Disclosure Statement
No competing financial interests exist.
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44
Table 1 (Desarrollo 1). Nucleotide sequence of the primers used in multiplex polymerase
chain reaction for detection of 16S rRNA, mecA, and Panton-Valentine leukocidin genes
(McClure et al., 2006); and multilocus sequence typing analysis for detection of arcC, aroE,
glpF, gmk, pta, tpi, and yqiL genes (Enright et al., 2000).
Primer Oligonucleotide sequence Amplicon
Size (bp)
Staph 756 F 5’-AAC TCT GTT ATT AGG GAA GAA CA-3’
Staph 750 R 5’-CCA CCT TCC TCC GGT TTG TCA CC-3’ 756
mecA 1 F 5’-GTA GAA ATG ACT GAA CGT CCG ATA A-3’
mecA-2 R 5’-CCA ATT CCA CAT TGT TTC GGT CTA A-3’ 310
luk-PV-1 F 5’-ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A-3’
luk-PV-2 R 5’-GCA TCA AGT GTA TTG GAT AGC AAA AGC-3’ 433
arcC F 5'-TTG ATT CAC CAG CGC GTA TTG TC-3'
arcC R 5'-AGG TAT CTG CTT CAA TCA GCG-3' 456
aroE F 5'-ATC GGA AAT CCT ATT TCA CAT TC-3'
aroE R 5'-GGT GTT GTA TTA ATA ACG ATA TC-3' 456
glpF F 5'-CTA GGA ACT GCA ATC TTA ATC C-3'
glpF R 5'-TGG TAA AAT CGC ATG TCC AAT TC-3' 465
gmK F 5'-ATC GTT TTA TCG GGA CCA TC-3'
gmK R 5'-TCA TTA ACT ACA ACG TAA TCG TA-3' 429
pta F 5'-GTT AAA ATC GTA TTA CCT GAA GG-3'
pta R 5'-GAC CCT TTT GTT GAA AAG CTT AA-3' 474
tpi F 5'-TCG TTC ATT CTG AAC GTC GTG AA3'
tpi R 5'-TTT GCA CCT TCT AAC AAT TGT AC-3' 402
yqiL F 5'-CAG CAT ACA GGA CAC CTA TTG GC-3'
yqiL R 5'-CGT TGA GGA ATC GAT ACT GGA AC-3' 516
45
Table 2 (Desarrollo 1). Identification of 16S rRNA, mecA and Panton-Valentine leukocidin
(PVL) genes in Staphylococcus aureus and methicillin-resistant Staphylococcus aureus
isolates from animals and retail meat.
No. of isolates with the specific gene (%) Source
No. of
samples
No. of samples positive
for S. aureus (%) 16S rRNA mecA PVL
Animal
Sheep
Pig
Cow
Total
Raw meat
Pork
Chicken
Beef
Total
Deli meat
Ham
Turkey
Chicken
Total
64
60
43
167
71
37
37
145
21
16
9
46
26 (40.6)
30 (50.0)
2 (4.7)
58 (34.7)
35 (49.3)
25 (67.6)
10 (27.0)
69 (47.6)
4 (19.0)
0 (0.0)
2 (22.2)
6 (13.0)
26 (40.6)
30 (50.0)
2 (4.7)
58 (34.7)
35 (49.3)
25 (67.6)
10 (27.0)
69 (47.6)
4 (19.0)
0 (0.0)
2 (22.2)
6 (13.0)
0 (0.0)
5 (7.0)
5 (3.4)
0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)
46
Table 3 (Desarrollo 1). Antimicrobial resistance of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) isolates from
animals and retail meat.
Antimicrobial No. (%) of resistant all S. aureus isolates No. (%) of resistant
MRSA isolates
Subclass Agent Animal (n=58)
(Sheep; Pig; Cow) Raw meat (n=69)
(Pork; Chicken; Beef) Deli meat (n=6)
(Ham;Turkey;Chicken) Pork meat
(n=5) Macrolides Tetracyclines Fluoroquinolones Phenicols Penicillins Aminoglycosides Streptogramin Lincosamides
Erythromycin Tetracycline Ciprofloxacin Chloramphenicol Penicillin Gentamicin Kanamycin Streptomycin Quinupristin/dalfopristin Lincomycin
3 (5.2) (0; 3; 0)
47 (81.0) (24; 23; 0)
3 (5.2) (0; 3; 0)
49 (84.5) (19; 30; 0)
1 (1.7) (1; 0; 0)
6 (10.3) (1; 5; 0)
38 (65.5) (13; 24; 1)
28 (40.6) (25; 3; 0)
29 (42.0) (27; 2; 0)
2 (2.9) (2; 0; 0)
2 (2.9) (2; 0; 0)
35 (50.7) (27; 4; 4)
1 (1.4) (1; 0; 0)
2 (2.9) (2; 0; 0)
2 (2.9) (2; 0; 0)
29 (42.0) (26; 3; 0)
1 (16.7) (0; 0; 1)
2 (33.3) (2; 0; 0)
1 (16.7) (1; 0; 0)
4 (80.0)
4 (80.0)
5 (100.0)
1 (20.0)
4 (80.0)
The following antimicrobials were tested using the National Antimicrobial Resistance Monitoring System (NARMS) panel: tigecycline (range 0.015-0.5 µg/ml); tetracycline (1-32); chloramphenicol (2-32); daptomycin (0.25-16); streptomycin (512-2048); tylosin tartrate (0.25-32); quinupristin/dalfopristin (0.5-32); linezoid (0.5-8); nitrofutantoin (2-64); penicillin (0.25-16); kanamycın (128-1024); erythromycin (0.25-8); ciprofloxacin (0.12-4); vancomycin (0.25-32); lincomycin (1-8); gentamicin (128-1024). All isolates were susceptible to vancomycin, tigecycline, daptomycin, tylosin tartrate, nitrofurantoin and linezolid.
M 1 2 3 4 5 6 7 8 9 10 M
47
Table 4 (Desarrollo 1). Minimum inhibitory concentrations (MICs) of resistant Staphylococcus aureus and methicillin-resistant S.
aureus isolates from animals and retail meat.
No. (%) of resistant S. aureus isolates with MIC (µg/mL) of Antimicrobial Agent (breakpoints) Total
0.5 - 1 2 4 >4 8 >8 16 >16 32 >32 256 >256 Erythromycin (≥8 µg/mL)a 32 32
(100.0)
Tetracycline (≥16 µg/mL)a 76 11 (14.5)
15 (19.7)
50 (65.8)
Ciprofloxacin (≥4 µg/mL)a 2 2 (100.0)
Chloramphenicol (≥32 µg/mL)a 5 3 (60.0)
2 (40.0)
Penicillin (≥0.25 µg/mL)a 86 41 (47.7)
9 (10.5)
14 (16.3)
10 (11.6)
7 ( 8.1)
5 (5.8)
Gentamicin (≥16 µg/mL)a 2 2 (100.0)
Kanamycin (≥64 µg/mL)a 2 2 (100.0)
Streptomycin (≥8 µg/mL)b 6 6 (100.0)
Quinupristin/dalfopristin (≥8 µg/mL)a 2 1 (50.0)
1 (50.0)
Lincomycin (≥4 µg/mL)c 68 4 ( 5.6)
3 ( 4.4)
61 (89.7)
aLevels of MIC values against tested antibiotics (CLSI, 2009). bLevels of MIC values against tested antibiotics (Jarløv et al., 1997). cLevels of MIC values against tested antibiotics (Nemati et al., 2008).
48
Table 5 (Desarrollo 1). Antimicrobial resistance profiles of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA)
isolates from animals and retail meat.
No. (%) of all S. aureus isolates with the specific profile
No. (%) of MRSA isolates with the specific profile Antimicrobial resistance profile
No. of antimicrobial subclasses resistant to
Animal (n=58)
Raw meat (n=69)
Deli meat (n=6)
Raw meat (n=5)
ERY-PEN-TET-LINC-CHL-GEN-CIP-QUI/DAL 8 1 ( 1.4) ERY-PEN-TET-LINC-CHL-CIP-QUI/DAL 7 1 ( 1.4) ERY-PEN-TET-LINC-CHL-STR 6 2 ( 3.4) ERY-PEN-TET-LINC-KAN 5 1 ( 1.4) PEN-TET-LINC-CHL-STR 5 1 ( 1.7) PEN-TET-LINC-GEN 4 1 ( 1.7) PEN-TET-LINC-KAN 4 1 ( 1.4) 1 (20.0) PEN-TET-LINC-STR 4 2 ( 3.4) ERY-PEN-TET-LINC 4 1 ( 1.7) 13 (18.8) 3 (60.0) PEN-TET-LINC 3 22 (37.9) 1 ( 1.4) PEN-LINC-STR 3 1 ( 1.7) ERY-PEN-LINC 3 2 ( 2.9) ERY-TET-LINC 3 5 ( 7.2) PEN-LINC 2 4 ( 6.9) 1 ( 1.4) 1 (16.7) PEN-TET 2 12 (20.7) 2 ( 2.9) TET-LINC 2 3 ( 5.2) ERY-LINC 2 3 ( 4.3) ERY-PEN 2 2 ( 2.9) 1 (20.0) LINC 1 1 ( 1.7) PEN 1 3 ( 5.2) 10 (14.5) 1 (16.7) TET 1 3 ( 5.2) 4 ( 5.8) ERY 1 1 (16.7) Susceptible to all tested 0 2 ( 3.4) 22 (31.9) 3 (50.0)
CIP, ciprofloxacin; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; KAN, kanamycin; LINC, lincomycin; QUI/DAL, quinupristin/dalfopristin; PEN, penicillin; STR, streptomycin, TET, tetracycline.
M 1 2 3 4 5 6 7 8 9 10 M
49
Figure 1 (Desarrollo 1). Dendrogram showing the genetic relatedness of 100 Staphylococcus
aureus isolates as determined by the analysis of pulsed-field gel electrophoresis (PFGE)
profiles by the unweighted-pair group method with arithmetic averages with the sequence type
(ST). The scale indicates levels of similarity within this set of isolates. Red line indicates the
cutoff at 80%. Numbers represent the samples codes, followed on the right by the STs and the
type of the sample. Clusters are separated by horizontal lines and labeled with correlative
numbers. Asterisks indicate methicillin-resistant Staphylococcus aureus isolates determined
by multiplex polymerase chain reaction targeting the mecA gene.
50
PFGE SmaI
100
9590858075706560555045
PFGE SmaI
118
120
92
52
53
27
87
91
72
73
113
98
99
88
111
93
108
110
11
89
10
112
70
119
121
123
102
103
85
84
82
83
97
114
115
116
117
95
100
101
105
106
107
96
104
86
94
129
130
131
133
134
125
126
127
128
22
135
136
137
138
139
140
141
142
143
144
124
1
14
57
63
61
3
5
4
66
67
65
60
18
145
78
79
80
81
77
62
64
76
122
49
109
15
7
132
23
32
36
37
39
30
NT
398
50
398
508
72
1
1
1
6
6
6
188
1159
1159
1159
2187
2187
352
5
5
146
5
5
5
5
5
5
5
5
NT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
8
9
9
9
9
9
9
9
9
9
5
5
5
5
5
15
15
15
15
15
15
133
133
133
2111
133
133
2111
Deli Meat Chicken
Deli Meat Ham
Bovine Meat
Porcine Meat
Porcine Meat
Ovine
Poultry Meat
Bovine Meat
Porcine Meat
Porcine Meat
Bovine Meat
Poultry Meat
Poultry Meat
Poultry Meat
Bovine Meat
Bovine Meat
Bovine Meat
Bovine Meat
Bovine
Bovine Meat
Bovine
Bovine Meat
Porcine Meat
Deli Meat Ham
Deli Meat Chicken
Deli Meat Ham
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Poultry Meat
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine
Porcine Meat
Porcine Meat
Porcine Meat
Porcine
Porcine
Porcine
Porcine Meat
Porcine Meat
Porcine Meat
Porcine Meat
Porcine
Porcine
Porcine Meat
Porcine Meat
Porcine Meat
Porcine Meat
Porcine Meat
Porcine Meat
Porcine Meat
Porcine Meat
Deli Meat Ham
Porcine
Bovine Meat
Ovine
Ovine
Ovine
Ovine
Ovine
Ovine
Ovine
1
2
3
4
5
6
7
* *
Isolate # S Meat/Anima
51
2. Multiplex real-time PCR for detection of Staphylococcus aureus, mecA
and Panton-Valentine Leukocidin (PVL) genes from selective
enrichments from animals and retail meat
Valeria Velasco1,2, Julie S. Sherwood1, Pedro Rojas3, Catherine M. Logue4*
1Department of Veterinary and Microbiological Sciences, North Dakota State University,
Fargo, North Dakota, United States of America, 2Department of Animal Sciences, University of Concepción, Chillán, Chile, 3Laboratory of Animal Physiology and Endocrinology, Veterinary Sciences, University of
Concepción, Chillán, Chile, 4Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary
Medicine, Iowa State University, Ames, Iowa, United States of America
Enviado a PLOS ONE, en proceso de revisión.
Abstract
The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR
method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals
and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were
examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-
time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with
methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment
samples were assessed. The conventional culture/PCR method included the two-step selective
enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The
conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of
real-time PCR on samples following primary and secondary enrichment detected S. aureus in
111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was
0.68-0.88 (from substantial to almost perfect agreement) and 0.29-0.77 (from fair to
substantial agreement) for primary and secondary enrichments, respectively, using real-time
PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond
52
that expected by chance to moderate agreement) for primary and secondary enrichment
samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay
detected the mecA gene in some samples that were negative for S. aureus, but positive for
Staphylococcus spp. The PVL gene was not detected in any sample by the conventional
culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional
culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in
animals and raw meat samples. The real-time PCR assay may be recommended as a rapid
method for the detection of S. aureus and the mecA gene, with further confirmation of
methicillin-resistant S. aureus (MRSA) using the standard culture method.
*Corresponding Author
E-mail: [email protected]
1802 University Blvd, VMRI #2
Iowa State University, Ames, IA 50011, USA
Ph 515 294 3785; fax 515 294 1401
2.1 Introduction
Staphylococcus aureus is considered as an important cause of a wide variety of diseases in
humans such as: food poisoning, pneumonia, wound and nosocomial infections [1,2]. There
are many anti-staphylococcal agents; however, the bacterium has developed mechanisms to
neutralize them such as the methicillin resistance mechanism [3]. Methicillin-resistant S.
aureus (MRSA) is an increasing cause of health care-associated (HA-MRSA) [1], community-
associated (CA-MRSA) [2], and livestock-associated (LA-MRSA) infections worldwide [4].
The altered penicillin-binding protein (PBP2a) is associated with methicillin resistance. This
protein has a reduced affinity for β-lactam antibiotics [5,6], and is encoded by the mecA gene,
which is carried on the staphylococcal cassette chromosome mec (SCCmec) [5]. CA-MRSA
strains are more likely to encode the Panton–Valentine leukocidin (PVL) toxin, which is a
pore-forming toxin considered as a virulence factor [7,8]. The PVL toxin has been related to
life-threatening CA-MRSA infections and deaths, primarily severe skin infections and tissue
necrosis [9].
53
In the United States, approximately 29% (78.9 million people) and 1.5% (4.1 million) of the
population were estimated to be nasal carriers of S. aureus and MRSA, respectively [10]. An
estimated 478,000 hospitalizations corresponded to S. aureus infections, of which 278,000
hospitalizations were attributed to MRSA infections in 2005 [11]. In addition, the carriage of
MRSA in meat-producing animals [12-14] and the contamination of retail meat with MRSA
[15-17] have increased the concern that food may serve as a vehicle to transmit MRSA to the
human population [17].
Different culture methods have been used to detect MRSA. Generally, conventional
microbiological procedures are laborious, since they require the isolation of S. aureus before
assessing methicillin resistance. However, culture methods are still considered as standard
methods for traditional confirmation of S. aureus. Wertheim et al. (2001) [18] developed a
selective media containing phenol red and antibiotics (aztreonam and ceftizoxime), increasing
the sensitivity of the detection of MRSA after 48 h of incubation, but at the expense of longer
time needed for confirmation. The isolation and identification of MRSA, including selective
enrichment and plating on selective agars, followed by confirmation using biochemical testing
and/or PCR assays, requires 3-7 days approximately [15,16,19]. Therefore, development of a
rapid method for detection of MRSA has become an important need in the microbiological
analysis of samples especially those where there is a potential risk of exposure for humans.
Real-time PCR technology has been used as an alternative to culture methods for the rapid
detection of S. aureus and MRSA. Detection using real-time PCR may decrease the time of
analysis to 18 h after consecutive broth enrichment in clinical samples [20]; or <2 h in positive
blood cultures [21,22]. However, most studies have used real-time PCR to detect MRSA in
clinical samples and isolates and a few studies have evaluated the application of this method
for the detection of MRSA in animals [23,24] and meat [15,25,26].
Since S. aureus and MRSA have been found in food-producing animals and retail meat,
increasing the concern about the exposure for humans through the food chain, and there is a
need to decrease the time of analysis, we analyzed samples obtained from animals and retail
meats using primary and secondary selective enrichments in order to detect nuc (identification
of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor) genes
54
using a multiplex real-time PCR assay. The results obtained with the real-time PCR assay
were compared with the results from a culture method, considered as the standard method,
which also included the two-step selective enrichment, followed by selective plating,
biochemical testing and conventional multiplex PCR. Positive samples obtained with the
culture method were characterized by multilocus sequence typing (MLST) and the
antimicrobial resistance profiles were obtained.
2.2 Materials and Methods
2.2.1 Samples
A total of 77 nasal swabs (Becton, Dickinson and Company, Sparks, MD, USA) were
collected from animals (sheep, n=35; pigs, n=28; cows, n=14) sampled immediately after
stunning at the Meat Lab (Department of Animal Sciences); and at the ND Veterinary
Diagnostic Lab at North Dakota State University, Fargo, ND. Animal samples were collected
during the period May 2010-April 2011. The protocol of sampling was approved by the North
Dakota State University Institutional Biosafety Committee (B10014).
In addition, 112 retail raw meat (pork, n=39; chicken, n=37; beef, n=36) and 45 deli meat
(ham, n=20; turkey, n=16; chicken, n=9) samples were randomly purchased from four
different supermarket chains in Fargo, ND. Sampling visits were made between June 2010 and
January 2011. All samples were immediately stored at 4°C and processed within six hours of
collection.
2.2.2 Culture method
Staphylococcus aureus were isolated by the two-step selective enrichment procedure
according to the method described by de Boer et al. (2009) [15] followed by plating steps on
selective agar. Briefly, for the primary enrichment, a 25 g sample of retail meat and 225 mL of
MHB+6.5%NaCl (Mueller-Hinton broth [Difco, Becton, Dickinson, Sparks, MD, USA] with
added 6.5% sodium chloride [VWR International, West Chester, PA, USA]) were placed in a
sterile stomacher bag and homogenized using a stomacher®400 circulator (Seaward, England)
at 230 rpm for 90 seconds. The suspension was incubated for 18-20 h at 37ºC. Following
55
primary enrichment, a secondary enrichment was used by inoculating 1 mL of the primary
enrichment broth into 9 mL of PHMB+ (D-mannitol in phenol red mannitol broth base [Difco,
Becton, Dickinson, Sparks, MD, USA] containing ceftizoxime [5 µg mL-1, US Pharmacopeia,
Rockville, MD, USA] and aztreonam [75 µg mL-1, Sigma Chemical CO., Louis, MO, USA]
according to Wertheim et al. [2001] [18]), followed by incubation for 18-20 h at 37ºC. Nasal
swabs from animals were placed directly in 9 mL MHB+6.5%NaCl and incubated for 18-20 h
at 37ºC. Then, the secondary enrichment was used following the procedure described above.
Following incubation of the secondary enrichment broth, all samples were struck directly to
BP medium (Baird-Parker medium [Difco, Becton, Dickinson, Sparks, MD, USA])
supplemented with egg yolk tellurite according to manufacturer’s recommendations and
incubated for 48 h at 37°C. Presumptive S. aureus colonies (black colonies surrounded by 2 to
5 mm clear zones) were transferred to TSA II 5%SB plates (Trypticase soy agar with 5%
sheep blood [Difco, Becton, Dickinson, Sparks, MD, USA]) and incubated for 18-20 h at 37ºC.
Suspect S. aureus colonies (presence of β-haemolysis) were confirmed using Sensititre Gram
Positive ID (GPID) plates (Sensititre®, TREK Diagnostic Systems Ltd., Cleveland, OH, USA)
according to the manufacturer’s instructions.
2.2.3 Conventional multiplex PCR method
Confirmed S. aureus strains were recovered from frozen stock to TSA plates (Trypticase soy
agar [Difco, Becton, Dickinson, Sparks, MD, USA]) and incubated at 37°C for 18-24 h. DNA
extraction was carried out by suspending one colony in 50 µL of DNase/RNase-free distilled
water (Gibco Invitrogen, Grand Island, NY, USA), heating (99°C, 10 min) and centrifugation
(30,000×g, 1 min) to remove cellular debris. The remaining DNA was transferred to a new
tube and stored at -20°C.
A multiplex PCR assay for the detection of 16S rRNA (identification of S. aureus), mecA
(associated with methicillin resistance) and PVL-encoding genes (virulence factor) (Table 1)
included 2 µL of the DNA template (described above) added to a 50 µL final reaction mixture
containing: 1X Go Taq® Reaction Buffer (pH 8.5), 0.025 U µL-1 of Go Taq® DNA
56
polymerase, 200 µM dNTP (Promega, Madison, WI, USA) and 1 µM of primers (16S rRNA,
mecA, LukS/F-PV) (Integrated DNA Technologies, Inc., Coralville, IA, USA).
Multiplex PCR reactions were carried out in a thermocycler (Eppendorf, Hamburg, Germany),
and the PCR conditions were adjusted according to the protocol described by Makgotlho et al.
(2009) [27] as follows: initial denaturation at 94°C for 10 min, followed by 10 cycles of
denaturation at 94°C for 45 s, annealing at 55°C for 45 s and extension at 72°C for 75 s
followed by another 25 cycles of 94°C for 45 s, 50°C for 45 s and a final extension step at
72°C for 10 min. An external positive control (DNA extracted from MRSA ATCC 35591,
positive for mecA and PVL genes) and an external negative control (DNase/RNase-free
distilled water) were included with each run.
PCR amplicons (10 µL) were loaded into a 1.5% (wt/vol) agarose gel (Agarose ITM) using
EzVision One loading dye (Amresco, Solon, OH, USA) and electrophoresis was carried out in
1X TAE buffer at 100 v for 1 h. A molecular weight marker 100-bp ladder (Promega, Madison,
WI, USA) were included on each gel. Bands were visualized using an Alpha Innotech UV
imager (FluorChemTM).
2.2.4 Multiplex real-time PCR assay
DNA was extracted from the primary and secondary enrichment broths of the animal and meat
samples using the boiling method described previously by de Medici et al. (2003) [28]. Five
microliters of DNA template extracted was used in the real-time iQTM Multiplex Powermix
(Bio-Rad Laboratories, Hercules, CA, USA), in a final volume of 20 µL per reaction.
The real-time PCR assay targeted the following genes: nuc (identification of S. aureus), mecA
(associated with methicillin resistance) and PVL-encoding genes (virulence factor) (Table 1).
The final concentrations in the reaction mixture were: 300 nM of primers (forward and
reverse), 200 nM of fluorogenic probes (Applied Biosystems, Foster City, CA, USA), and 1X
iQTM Multiplex Powermix (Bio-Rad Laboratories, Hercules, CA, USA), according to the
manufacturer's recommendations.
57
The thermal cycling conditions were adjusted to an initial denaturation of 3 min at 95 °C,
followed by 40 PCR cycles of 95 °C for 15 s and 55 °C for 1 min, using an iCycler IQTM real
time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). An external positive control
(DNA extracted from MRSA ATCC 35591, positive for mecA and PVL genes) and an external
negative control (DNase/RNase-free distilled water) were included with each plate. Data
analysis was carried out using the iCycler software version 3.0 (Bio-Rad Laboratories,
Hercules, CA, USA).
2.2.5 Characterization of S. aureus strains isolated by culture method
Multilocus Sequence typing (MLST)
Briefly, S. aureus isolates were struck to TSA plates and incubated at 37°C for 18–24 h.
Colonies were picked to 40 µL of single cell lysing buffer (50 µg/mL of Proteinase K,
Amresco; in TE buffer [pH=8]), and then lysed by heating to 80°C for 10 min followed by
55°C for 10 min in a thermocycler. The final suspension was diluted 1:2 in sterile water,
centrifuged to remove cellular debris, and transferred to a sterile tube (Marmur, 1961) [29].
The housekeeping genes: arcC, aroE, glpF, gmk, pta, tpi, and yqiL, were amplified [30]. All
PCR reactions were carried out in 50-µL volumes: 1 µL of DNA template, Taq DNA
polymerase (Promega) (1.25 U), 1X PCR buffer (Promega), primers (0.1 µM) (Integrated
DNA Technologies, Inc.), and dNTPs (200 µM) (Promega). PCR settings were adjusted
according to Enright et al. (2000) [30] using a thermocycler (Eppendorf). Ten microliters of
the PCR products were loaded into 1% agarose gels in 1X TAE with EzVision One loading
dye, and run at 100V in 1X TAE for 1 h. Images were captured using an Alpha Innotech
imager. After PCR, each amplicon was purified of amplification primer using the
QIAquickPCR Purification Kit (Qiagen, Valencia, CA) as per manufacturer’s instructions.
Purified DNA was sequenced at Iowa State University’s DNA Facility (Ames, IA) using an
Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). Sequence
data were imported into DNAStar (Lasergene, Madison, WI), trimmed, and aligned to the
control sequences (from the MLST site) and interrogated against the MLST database
(http://saureus.mlst.net/). Sequence types were added to the strain information for analysis in
BioNumerics.
58
Resistance profiles
The antimicrobial resistance profiles (AR) of S. aureus isolates (n=95) were determined using
the broth microdilution method (CMV3AGPF, Sensititre, Trek Diagnostics), according to the
manufacturer’s and the National Antimicrobial Resistance Monitoring System (NARMS)
guidelines for animal isolates [31]. Antimicrobials in the panel and their resistance breakpoints
were as follows: erythromycin (≥8 µg/mL), tetracycline (≥16 µg/mL), ciprofloxacin (≥4
µg/mL), chloramphenicol (≥32 µg/mL), penicillin (≥16 µg/mL), daptomycin (no interpretative
criteria), vancomycin (≥32 µg/mL), nitrofurantoin (≥128 µg/mL), gentamicin (>500 µg/mL),
quinupristin/dalfopristin (≥4 µg/mL), linezolid (≥8 µg/mL), kanamycin (≥1024 µg/mL),
tylosin (≥32 µg/mL), tigecycline (no interpretative criteria), streptomycin (>1000 µg/mL), and
lincomycin (≥8 µg/mL). Resistance to at least three classes of antibiotics was considered as
multidrug resistance (MDR) [32].
2.2.6 Statistical analysis
The 95% confidence intervals for prevalence were obtained, using the plus four estimate when
positive or negative samples were less than 15. The Chi-square test was used to assess the
significance in proportion of positive samples between sample types, only if no more than
20% of the expected counts were less than 5 and all individual expected counts were 1 or
greater [33]. On the contrary, Fisher’s exact test was used with two-sided p-values. SAS
software version 9.2 (SAS Institute Inc., Cary, NC) was used to assess significance with a
P<0.05.
As there is no true gold standard method for S. aureus and MRSA detection, the kappa
statistic was calculated to compare agreement between real-time PCR assay (using primary
and secondary enrichment) and conventional culture/PCR method.
2.3 Results
The culture method included a biochemical identification to confirm S. aureus, which agreed
with the results of the conventional multiplex PCR that detected the gene 16S rRNA. This
method detected 95 positive S. aureus samples from a total of 234 samples collected (Table 2).
59
The multiplex real-time PCR assay using primary and a secondary enrichment samples,
recovered S. aureus (detection of nuc gene) from 111 and 120 samples of 234 samples
respectively.
By the conventional culture/PCR method alone, the rate of positive S. aureus samples was
found to be 41.6% (CI95%: 30.6-52.6%) in animals and 51.8% (CI95%: 42.5-61.0%) in raw
meat samples respectively; a significantly lower rate of 11.1% (CI95%: 4.5-24.1%) was
observed in deli meat (P≤0.05). Using the primary enrichment samples and real-time PCR, a
significantly higher recovery of S. aureus (P≤0.05) was found in animals (55.8%, CI95%:
44.8-66.9%) and raw meat (57.1%, CI95%: 47.9-66.3%) than in deli meat samples (8.9%,
CI95%: 3.1-21.4%). However, no significant difference (P>0.05) was found between the rate
of positive S. aureus samples in animals (53.2%, CI95%: 42.1-64.4%), raw meat (53.6%,
CI95%: 44.3-62.8%) and deli meat (42.2%, CI95%: 27.8-56.7%), when the secondary
enrichment samples were tested by real-time PCR. A significantly higher recovery of S.
aureus (P≤0.05) was obtained from deli meat when the secondary enrichment samples were
assessed by real-time PCR.
The mecA gene was detected in two pork samples (5.4%, CI95%: 0.7-18.8%) by the
conventional multiplex PCR preceded by the culture method, and by assessing the primary and
secondary enrichment samples by real-time PCR. The real-time PCR analysis detected the
mecA gene using both enrichments in samples that were negative by conventional multiplex
PCR in four pork meat and four deli meat samples. Using the primary enrichment, the real-
time PCR detected the mecA gene in one sample isolated from a sheep, and two from pork
meat, which were negative using the secondary enrichment. Using the secondary enrichment,
the real-time PCR detected the mecA gene from one sample isolated from a pig, two from pork
meat, and two from deli meat, which were negative using the primary enrichment.
The PVL gene was not detected in any sample by the conventional culture/PCR method or the
real-time PCR assay.
Table 3 shows the results of real-time PCR using primary and secondary enrichments on the
detection of S. aureus compared with a conventional culture/PCR method. Total agreement
60
and the kappa statistic for real-time PCR using the primary enrichment samples were 85.7%
(k=0.72, CI95%: 0.62-0.82), 83.9% (k=0.68, CI95%: 0.59-0.76), and 97.8% (k=0.88, CI95%:
0.78-0.97) for animals, raw meat, and deli meat respectively. For real-time PCR using the
secondary enrichment samples, the total agreement and the kappa statistic were 88.3% (k=0.77,
CI95%: 0.67-0.86), 87.5% (k=0.75, CI95%: 0.67-0.83), and 68.9% (k=0.29, CI95%: 0.16-
0.43) for animals, raw meat, and deli meat respectively. Positive agreement (sensitivity) was
100% for animal samples using both enrichments. For animals and raw meat, a higher
negative agreement (specificity) was obtained for real-time PCR using the secondary
enrichment.
Six samples isolated from animals and six from raw meat were deemed S. aureus negative by
the conventional culture/PCR method, but positive by real-time PCR using the primary and
secondary enrichments. Three S. aureus samples isolated from raw meat were positive by the
conventional culture/PCR method, but negative by the real-time PCR assay.
The real-time PCR method using the primary enrichment failed to detect the presence of S.
aureus in four samples: three isolated from raw meat (two from beef, one from poultry) and
one from deli meat (ham) that were positive by the culture method and by the real-time PCR
assay using the secondary enrichment samples. Using the secondary enrichment samples, the
real-time PCR assay failed to detect three samples isolated from raw meat (pork) that were S.
aureus positive by the culture method and using the primary enrichment in real-time PCR.
The results of real-time PCR using primary and secondary enrichment on the detection of the
mecA gene compared with a conventional culture/PCR method are shown in Table 4. Total
agreement for real-time PCR using the primary and secondary enrichment samples ranged
from 91.1% to 98.7% and from 86.7 to 98.7%, respectively. The kappa statistic was zero when
the mecA gene was not detected by the conventional culture/PCR method and 0.49 (CI95%:
0.39-0.58) for raw meat. Positive agreement (sensitivity) of 100% was obtained for raw meat
samples for both methods.
The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus
by the conventional culture/PCR method (one from a pig, one from a sheep, four from pork,
four from deli ham, and one from deli turkey). All of these samples were identified as
61
harboring S. epidermidis, S. saprophyticus or S. haemolyticus using biochemical analysis on
isolates recovered. However, three of these samples (one from a pig, two from pork meat)
tested positive for the nuc gene when primary and secondary enrichments were assessed by
real-time PCR.
Table 5 shows the antimicrobial resistance profiles and the sequence types of the ninety-five S.
aureus strains isolated from animals and retail meat by the conventional culture/PCR method.
A total of thirteen antimicrobial resistance profiles were identified among S. aureus isolates.
Most of the S. aureus isolates were resistant to tetracycline and lincomycin, and were of ST9.
A total of twenty-two S. aureus isolates exhibited multi-drug resistance. Susceptibility to all
antimicrobials tested were found in thirty-five S. aureus isolates, which were mostly recovered
from chicken meat and identified as ST5.
2.4 Discussion
In this study, a high recovery of S. aureus was found in animals and meat samples by the
culture/PCR method and the real-time PCR assay (Table 2). The inclusion of selective
enrichment steps has been found to increase the rate of detection of S. aureus [15]. Waters et
al. (2011) [26] also found a high prevalence of S. aureus in raw meat (47%) using a single step
selective enrichment protocol, followed by plating on Baird Parker agar, and confirmation by
real-time PCR targeting the femA gene.
The kappa statistic for detection of S. aureus using the primary enrichment in real-time PCR
was 0.68-0.88 (Table 3), which indicates a good agreement (substantial to almost perfect
agreement) with the conventional culture/PCR method. Using the secondary enrichment and
real-time PCR, the kappa statistic for detection of S. aureus was 0.29-0.77, resulting in a fair
agreement when deli meat was tested. This is due to the significantly higher recovery of S.
aureus from the secondary enrichment samples by real-time PCR (Table 2), and the lower
negative agreement (specificity) obtained with this method (Table 3). This observation
suggests that small numbers (or levels) of S. aureus could be missed when the primary
enrichment alone is used in real-time PCR, and that the recovery of potentially injured or non-
viable strains appears to be enhanced when a secondary enrichment is applied. The enhanced
detection also suggests that the use of a standard culture method or primary enrichment alone
62
could lead to higher false negative results. Therefore, including a secondary selective
enrichment step appears to improve the odds of detection of positive S. aureus samples.
Multiplex real-time PCR could detect more S. aureus positive samples than the conventional
culture/PCR method alone. Possible reasons for these discrepant results include: amplification
of DNA by the real-time PCR from very low levels of S. aureus that were not detectable by
the bacteriological methods due to competition or non-viable S. aureus in the samples, or
false-positive real-time PCR results as a result of cross-reaction rather than false-negative
culture results [23]. However, the possibility that these results are considered as false positives
in this study is probably very low, because the gene nuc, which was targeted by the real-time
PCR assay, has been used for specific detection and identification of S. aureus previously
[21,22,34,35]. Unfortunately, it was not possible to confirm these results by performing the
cultural method as detection was carried out from DNA extracts only, and the cells had
already been inactivated. The inability of real-time PCR to detect three S. aureus samples
isolated from raw meat that were positive by the culture method is somewhat unsatisfactory,
and could be considered as false-negative results.
For detection of mecA gene, the kappa statistic for both enrichments in real-time PCR was 0-
0.49 (Table 4). The k=0 indicates no agreement beyond that expected by chance, because the
real-time PCR assay detected the mecA gene probably from bacteria other than S. aureus and
the culture/PCR method detected the mecA gene from DNA extracted from confirmed S.
aureus strains. However, a few mecA positive samples were obtained from animals and meat
in this study (Table 2). Weese et al. (2010) [25] detected a low prevalence of MRSA in
samples isolated from retail meat (9.6% in pork, 5.6% in beef and 1.2% in chicken), using a
single-step selective enrichment protocol, followed of plating and biochemical testing.
The detection of the mecA gene by the real-time PCR assay in samples that were negative for
S. aureus by the conventional culture/PCR method may be due to the fact that either
coagulase-negative staphylococci and non S. aureus species can also carry the mecA gene
[21,36-38]. In this study, such samples were identified as Staphylococcus spp. positive by
biochemical testing. In addition, the mecA gene has been found in non-staphylococcal genera,
such as: Proteus vulgaris, Morganella morganii, Enterococcus faecalis [39] suggesting that its
63
use in a rapid screening technique would need further validation to avoid false-positive MRSA
data being generated. In this study, the DNA extraction was carried out from selective
enrichments, which could contain DNA from coagulase-positive or coagulase-negative
staphylococci or non-staphylococcal species that may carry the mecA gene, therefore a
positive result for the nuc and mecA genes does not indicate the presence of S. aureus carrying
the mecA gene.
None of the samples obtained from animals and retail meat were positive for the PVL genes
using both methods the conventional multiplex PCR and the real-time PCR. A similar
observation was reported by Weese et al. (2010) [25], who also failed to detect PVL positive
samples in raw meat in Canada using the real-time PCR technique. The PVL genes encode the
Panton-Valentine leukocidin toxin, which is a virulence factor that have been found in severe
cases of CA-MRSA [7,8,9].
Decreasing the time of detection of S. aureus and MRSA has become an important goal in
microbiological analysis of clinical samples. However, since S. aureus ST398, multi-drug
resistant S. aureus (Table 5), and MRSA are present in animals and meat [12-17], decreasing
the time of analysis may allow for prompt action to take place thus reducing the spread of
those strains in the food chain. The real-time PCR assay can potentially decrease the total time
for detection of S. aureus and the presence of the mecA gene in animal and meat samples.
Using the two-step selective enrichment the total time was <2 days by the real-time PCR
method, compared with a total time of 6-7 days using the culture method that includes
selective enrichments, plating steps, biochemical testing and a conventional multiplex PCR for
confirmation. However, the presence of MRSA should be confirmed by a culture method if
isolates are required for follow on studies. Some real-time PCR assays have been developed
for the rapid detection of MRSA from clinical samples [37,40-42]. Danial et al. (2011) [42]
reported that the real-time PCR assay detected 0.7% more MRSA-positive samples than the
routine standard Brilliance Chromogenic MRSA agar culture method in a total time of 8 h.
Huletsky et al. (2004) [40] detected MRSA directly from clinical specimens containing a
mixture of staphylococci in less than 1 h, with a false-positive detection rate of 4.6% for
MRSA that was actually MSSA. Paule et al. (2005) [41] developed a multiplex real-time PCR
64
that detected the genes femA and mecA directly from blood culture bottles in 2-3 h, obtaining
an indeterminate rate of 0.9% when coagulase-negative staphylococci strains were included.
In conclusion, the application of real-time PCR using selective enrichments appears to
improve the detection of S. aureus and the mecA gene in samples extracted from animals, raw
meat and deli meat. The real-time PCR assay may be recommended as a rapid method to
detect S. aureus and the mecA gene in samples obtained from the meat production chain;
however, if further confirmation of MRSA should be required (isolate recovery) then the
application of the standard culture method in parallel may be warranted.
Acknowledgements
The authors thank the staff of the Meat Lab (Department of Animal Sciences) and the ND
Veterinary Diagnostic Lab at North Dakota State University for assistance with the sample
collection. This study was funded through the Dean's Office, College of Agriculture, North
Dakota State University and The Dean's Office, College of Veterinary Medicine, Iowa State
University. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Conflict of interest
No conflict of interest declared.
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Table 1 (Desarrollo 2). Nucleotide sequence of the primers and probes used in conventional
multiplex PCR, multiplex real-time PCR.
Primer or probe name
Sequence (5'→3') 5' Reporter dye
3' Quencher 16S rRNA* Staph-756F AAC TCT GTT ATT AGG GAA GAA CA Staph-750R CCA CCT TCC TCC GGT TTG TCA CC
nuc†
nuc For CAA AGC ATC AAA AAG GTG TAG AGA nuc Rev TTC AAT TTT CTT TGC ATT TTC TAC CA Texas Red
nuc Probe TTT TCG TAA ATG CAC TTG CTT CAG GAC CA Iowa Black
mecA mecA-1F* GTA GAA ATG ACT GAA CGT CCG ATA A mecA-2F* CCA ATT CCA CAT TGT TTC GGT CTA A mecA For† GGC AAT ATT ACC GCA CCT CA mecA Rev† GTC TGC CAC TTT CTC CTT GT FAM†
mecA Probe† AGA TCT TAT GCA AAC TTA ATT GGC AAA TCC TAMRA†
PVL luk-PV-1F* ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A luk-PV-2R* GCA TCA AGT GTA TTG GAT AGC AAA AGC PVL For† ACA CAC TAT GGC AAT AGT TAT TT PVL Rev† AAA GCA ATG CAA TTG ATG TA Cy5†
PVL Probe† ATT TGT AAA CAG AAA TTA CAC AGT TAA ATA TGA Iowa Black
*Conventional multiplex PCR, according to McClure et al. (2006) [43]. †Multiplex real-time PCR, according to McDonald et al. (2005) [35].
69
Table 2 (Desarrollo 2). Detection of S. aureus, mecA and PVL genes from animals and retail
meat using a conventional culture/PCR method and a real-time PCR assay.
Real-time PCR Culture/PCR method
(No. of positives) Primary enrichment
(No. of positives)
Secondary enrichment
(No. of positives) Sample
type
No. of
samples S.aureu
s
mec
A PVL
S.aureu
s
mec
A PVL
S.aureu
s
mec
A PVL
Animals
Cow 14 0 0 0 4 0 0 3 0 0
Pig 28 21 0 0 25 0 0 24 1 0
Sheep 35 11 0 0 14 1 0 14 0 0
Total 77 32 0 0 43 1 0 41 1 0
Meat
Beef 36 9 0 0 10 0 0 12 0 0
Pork 37 25 2 0 26 6 0 27 6 0
Poultry 39 24 0 0 28 0 0 21 0 0
Total 112 58 2 0 64 6 0 60 6 0
Deli meat
Chicken 9 2 0 0 2 0 0 4 0 0
Ham 20 3 0 0 2 3 0 11 5 0
Turkey 16 0 0 0 0 1 0 4 1 0
Total 45 5 0 0 4 4 0 19 6 0
Total 234 95 2 0 111 11 0 120 13 0
70
Table 3 (Desarrollo 2). Raw agreement indices among conventional culture/PCR method and
real-time PCR assay, with two-step enrichment procedure for detection of S. aureus from
animals and retail meat.
No. (%) of samples* Comparison
within each
sample type
No. of
samples
No. positive
by
culture/PCR
method
Positive
agreement
(Sensitivity)
Negative
agreement
(Specificity)
Total
agreement
kappa
statistic
Real-time PCR
primary
enrichment
Animals 77 32 32 (100.0) 34 (75.6) 66 (85.7) 0.72
Meat 112 58 52 (89.7) 42 (77.8) 94 (83.9) 0.68
Deli meat 45 5 4 (80.0) 40 (100.0) 44 (97.8) 0.88
Real-time PCR
secondary
enrichment
Animals 77 32 32 (100.0) 36 (80.0) 68 (88.3) 0.77
Meat 112 58 52 (89.7) 46 (85.2) 98 (87.5) 0.75
Deli meat 45 5 5 (100.0) 26 (65.0) 31 (68.9) 0.29
*Percentages for positive agreement with culture/PCR method number positive as the denominator. Percentages for negative agreement with culture/PCR method number negative as the denominator. Percentage total agreement is obtained from the sum of the positive and negative agreement frequencies divided by the total sample size within each sample type.
71
Table 4 (Desarrollo 2). Raw agreement indices among conventional culture/PCR method and
real-time PCR assay, with two-step enrichment procedure for detection of the mecA gene from
animals and retail meat.
No. (%) of
samples*
Comparison
within each
sample type
No. of
samples
No. positive
by
culture/PCR
method
Positive
agreement
(Sensitivity)
Negative
agreement
(Specificity)
Total
agreement
kappa
statistic
Real-time PCR
primary
enrichment
Animals 77 0 - 76 (98.7) 76 (98.7) 0.00
Meat 112 2 2 (100.0) 106 (96.4) 108 (96.4) 0.49
Deli meat 45 0 - 41 (91.1) 41 (91.1) 0.00
Real-time PCR
secondary
enrichment
Animals 77 0 - 76 (98.7) 76 (98.7) 0.00
Meat 112 2 2 (100.0) 106 (96.4) 108 (96.4) 0.49
Deli meat 45 0 - 39 (86.7) 39 (86.7) 0.00
*Percentages for positive agreement with culture/PCR method number positive as the denominator. Percentages for negative agreement with culture/PCR method number negative as the denominator. Percentage total agreement is obtained from the sum of the positive and negative agreement frequencies divided by the total sample size within each sample type.
72
Table 5 (Desarrollo 2). Antimicrobial resistance profiles and sequence types of S. aureus
isolated by conventional culture/PCR method from animals and retail meat.
Antimicrobial resistance profile* No. of antimicrobial subclasses
No. of S. aureus isolates with the specific profile
Sequence types (n) †
PEN-TET-ERY-TYL-LINC-STR-CHL 6 2 Pig-ST9 (2) PEN-TET-LINC-STR-CHL 5 1 Pig-ST9 (1) TET-ERY-TYL-LINC 3 7 Pork-ST398 (5) Pork-ST5**
Pork-ST9 (1) PEN-LINC-STR 4 1 Pig-ST9 (1) PEN-ERY-LINC 3 7 Pork-ST9 (4) Pork-ST15 (2)
Pork-ST8 (1) TET-LINC-STR 3 1 Pig-ST9 ( 1) ERY-TYL-LINC 2 3 Chicken-ST5 (3) PEN 1 3 Pork-ST5 (1) Pork -ST5 (1)*
Pork-ST9 (1) TET 1 13 Sheep-ST398 (3) Sheep-ST133 (2)
Sheep-ST2111 (1) Pig-ST9 (1) Pork-ST1 (2) Pork-ST5 (2) Pork-ST398 (1) Pork-ST15 (1)
ERY 1 1 Deli chickenChicken-ST39 (1) LINC 1 5 Pig-ST9 (3) Sheep-ST133 (1)
Deli ham-ST15 Susceptible to all tested 0 3525 Chicken-ST5 (1513) Chicken-ST6
(32) Chicken-ST508 (1) Chicken-NT‡ (1) Pork-ST5 (2) Beef-ST1159 (3) Beef-ST2187 (1) Beef-ST188 (1) Beef-ST15 (1) Beef-ST72 (1) Beef-ST5 (1) Beef-ST1 (1)Deli ham-ST146 (1) Deli ham-ST5 (1) Deli chicken-ST5 (1) Pig-ST9 (1)
Total 95
*Antimicrobial abbreviations are as following: CHL, chloramphenicol; ERY, erythromycin; LINC, lincomycin; PEN, penicillin; STR, streptomycin, TET, tetracycline, TYL, tylosin. †ST, sequence type. ‡NT, non-typeable. *mecA gene positive.
73
3. Staphylococcus aureus harboring mecA gene and genes encoding
Panton-Valentine Leukocidin (PVL) exotoxin from humans
Valeria Velasco1,2, Esra Buyukcangaz3, Julie S. Sherwood1, Ryan M. Stepan1, Ryan J.
Koslofsky1 and Catherine M. Logue4
1 Department of Veterinary and Microbiological Sciences, North Dakota State University,
Fargo, ND, 58105, USA
2 Department of Animal Sciences, University of Concepción, Chillán, Chile
3 Laboratory of Animal Physiology and Endocrinology, Veterinary Sciences, University of
Concepción, Chillán, Chile
4 Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary
Medicine, Iowa State University, Ames, IA, 50011, USA
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) harbors the mecA gene, which encodes
the low-affinity penicillin-binding protein 2a (PBP2a), and most community-associated
MRSA (CA-MRSA) strains harbor genes encoding the Panton–Valentine leukocidin (PVL)
exotoxin. The aim of this study was to determine the occurrence of S. aureus harboring mecA
and PVL genes in hospital patients and healthy people, in North Dakota, United States. A total
of 108 MRSA isolates recovered from hospital patients were examined. Nasal swabs were
taken from 550 healthy subjects, and S. aureus was isolated and identified by culture method
and biochemical testing. Multiplex PCR (mPCR) was used to confirm S. aureus and to detect
the mecA and PVL genes. A total of 105 (97.2%) clinical isolates harbored the mecA gene and
11 (10.2%) PVL genes. The prevalence of nasal carriage of S. aureus in healthy people was
7.6%. None of the S. aureus strains obtained from healthy people were mecA- nor PVL-
positive. Therefore, Staphylococcus aureus harboring mecA and PVL genes is present in
patients affected by invasive infections, and the nasal carriage of S. aureus in healthy humans
seems to be low, without the presence of mecA and PVL genes, representing a low risk of
transmission among the community.
74
Keywords: Methicillin-resistant Staphylococcus aureus (MRSA), mecA, PVL, prevalence,
clinical isolates, healthy humans.
Address for Correspondence
Dr. Catherine M. Logue, Department of Veterinary Microbiology and Preventive Medicine,
1802 University Blvd, VMRI #2, College of Veterinary Medicine, Iowa State University,
Ames, IA 50011, USA. Ph 515 294 3785; Fax 515 297 1401. E-mail: [email protected]
3.1 Introduction
During the last decades, many bacterial species have developed resistance to antimicrobial
agents that have been commonly used to treat them (Swartz, 1997). Staphylococcus aureus is
one of the pathogens that are known to rapidly develop a resistance to antimicrobial agents
since new antibiotics are introduced (Lowy, 2003).
A few years after the introduction of penicillin for clinical use, the first penicillin-resistant S.
aureus was found. Methicillin-resistant S. aureus (MRSA) strains were identified in clinical
specimens in 1961, within one to two years of the introduction of methicillin (Jevons, 1961; de
Lencastre et al., 2007).
Methicillin-resistant S. aureus has been implicated in both community-associated (CA-
MRSA) and healthcare-associated (HA-MRSA) infections worldwide. Nasal carriage of S.
aureus and MRSA in humans in the United States was approximately 29% (78.9 million
people) and 1.5% (4.1 million people) respectively, in 2003-2004 (Gorwitz et al., 2008). In
2005, there were an estimated 478,000 hospitalizations that corresponded to S. aureus
infections, of which approximately 278,000 hospitalizations were attributed to MRSA
infections (Klein et al., 2007). In addition, an invasive MRSA infection was developed by
about 94,000 people, of which approximately 19,000 died. Of these infections, about 86%
were HA-MRSA and 14% were CA-MRSA (Klevens et al., 2007). However, HA-MRSA
clones have been progressively replaced by CA-MRSA strains due to the expanding
community reservoir and the increasing influx into the hospital of individuals who harbor CA-
MRSA (Benoit et al., 2008; D'Agata et al., 2009; Nimmo et al., 2013).
75
Resistance to methicillin in S. aureus is primarily mediated by the mecA gene, which encodes
the low-affinity penicillin-binding protein 2a (PBP2a) (Hartman and Tomasz, 1981; Van De
Griend et al., 2009). Most CA-MRSA strains harbor genes encoding the Panton–Valentine
leukocidin (PVL) exotoxin (Baba et al., 2002; Dufour et al., 2002), which has been related to
severe skin infections and necrotizing pneumonia (Ebert et al., 2009).
The objective of this study was to determine the occurrence of S. aureus harboring mecA and
PVL genes in hospital patients and healthy people, in North Dakota, United States.
3.2 Materials and Methods
3.2.1 Samples
A total of 108 MRSA isolates recovered from hospital patients with wound or blood stream
infections (sepsis, bone, cerobrospinal fluid [CSF], synovial fluid, subdural fluid, tissue, leg
ulcer and pleural fluid) were obtained from Bismarck State Hospital, Bismarck, ND, in July
2010.
In addition, during the Fall Semester 2010 and the Spring Semester 2011 a total of 550
samples were obtained from undergraduate students enrolled in the Department of Veterinary
and Microbiological Sciences, North Dakota State University (ND, USA), who were
considered as healthy people. Nasal swabs were taken from the subjects by using a sterile
moistened swab inserted into the nostril, to a depth of approximately 1 cm, and rotated five
times. For each subject, both nostrils were sampled using the same swab. Nasal swabs were
inoculated onto mannitol salt agar (MSA) plates (Becton, Dickinson and Company [BD],
Sparks, MD) and incubated at 37°C for 48 h. All colonies surrounded by yellow zones on
MSA after incubation were selected. Colonies with pink or red zones were excluded.
Presumptive S. aureus colonies in MSA were confirmed using Sensititre Gram Positive ID
(GPID) plates (Sensititre, TREK Diagnostic Systems Ltd., Cleveland, OH), according to the
manufacturer's recommendations.
MRSA clinical isolates and confirmed S. aureus isolates from healthy people were stored at -
80°C in brain–heart infusion broth (BD) containing 20% glycerol until use.
76
3.2.2 Multiplex polymerase chain reaction (mPCR)
All S. aureus strains were recovered from frozen stock to trypticase soy agar (TSA) plates
(BD) and incubated at 37°C for 18-24 h. DNA extraction was carried out by suspending one
colony in 50 µL of DNase/RNase-free distilled water (Gibco Invitrogen, Grand Island, NY,
USA), heating (99°C, 10 min) and centrifugation (30,000 × g, 1 min) to remove cellular debris.
The remaining DNA was transferred to a new tube and stored at -20°C.
Multiplex PCR assay for the detection of 16S rRNA (identification of S. aureus), mecA
(detection of MRSA) and PVL-encoding genes (Table 1) included 2 µL of the DNA template
(described above) added to a 50 µL final reaction mixture: 1X Go Taq® Reaction Buffer (pH
8.5), 0.025 U/µL of Go Taq® DNA polymerase, 200 µM dNTP (Promega, Madison, WI,
USA) and 1 µM of primers (16S rRNA, mecA, LukS/F-PV) (Integrated DNA Technologies,
Inc., Coralville, IA, USA).
Multiplex PCR reactions were carried out in a thermocycler (Eppendorf, Hamburg, Germany),
according to the protocol described by Makgotlho et al. (2009).
PCR amplicons (10 µL) were loaded into a 1.5% (wt/vol) agarose gel (Agarose ITM) using
EzVision One loading dye (Amresco, Solon, OH, USA) and electrophoresis was carried out in
1X TAE buffer at 100 V for 1 h. A molecular weight marker 100-bp ladder (Promega,
Madison, WI, USA) and a positive control (ATCC 35591) were included on each gel. Bands
were visualized using an Alpha Innotech UV imager (FluorChemTM).
3.2.3 Statistical analysis
The Chi-square test was used to assess the significance in prevalence of 16S rRNA, mecA, and
PVL-encoding genes in isolates between sample types, only if no more than 20% of the
expected counts were less than 5 and all individual expected counts were 1 or greater (Moore,
2007). On the contrary, Fisher’s exact test was used with a two-sided p-values. SAS software
version 9.2 (SAS Institute Inc., Cary, NC) was used to assess significance with a P<0.05.
77
3.3 Results
All clinical isolates were confirmed as S. aureus strains by the detection of 16S rRNA gene
using the mPCR technique. Previously, these clinical isolates had been identified as MRSA
strains in the hospital using microbiological procedures. Among the 108 MRSA clinical
isolates, a total of 105 (97.2%) harbored the mecA gene and 11 (10.2%) harbored PVL genes.
The mPCR method did not detect the PVL genes in MRSA strains isolated from hospital
patients affected by wound infections (Table 2).
The prevalence of nasal carriage of S. aureus in healthy people was 7.6%. There were not
significant differences (P≥0.05) between the seasons of sampling. None of these isolates
harbored the mecA gene nor PVL genes.
3.4 Discussion
Three clinical isolates identified as MRSA in the hospital did not harbor the mecA gene. This
may due to false positive results obtained by the microbiological method in the hospital or the
presence of the novel mecA homolog gene (mecALGA251 renamed as mecC) that has been
detected in MRSA isolated from humans and livestock that had been mecA-negative in recent
studies (García-Álvarez et al., 2011; Ito et al., 2012; Laurent et al., 2012; Monecke et al.,
2013; Petersen et al., 2013). The proportion of MRSA in relation to all S. aureus strains
causing infections is still unknown, difficulting the accurate estimations of the magnitude of
MRSA infections to take the right actions in public health policies (Klevens et al., 2007;
Moxnes et al., 2013).
In this study, the virulence factor PVL, that encodes the PVL exotoxin in S. aureus related to
severe skin infections and pneumonia, was detected solely in 11.1% of MRSA isolates from
patients affected by blood stream infections. MRSA isolates from patients affected by wound
infections did not harbor the PVL genes. Although the PVL genes were considered as a stable
marker for CA-MRSA, some CA-MRSA strains have been found to be PVL-negative (Nimmo
et al., 2013).
78
The isolation and identification of S. aureus strains in healthy people were carried out using a
culture procedure followed by a biochemical testing (Sensititre). The detection of S. aureus
positive colonies obtained by the biochemical testing agreed in 100% with the results obtained
by the mPCR method targetting the 16S rRNA gene. Therefore, both methods can be used for
confirmation of S. aureus strains.
In this study, the nasal carriage of S. aureus in healthy people was around 7.6%, which is
considerably lower than the prevalence found in other studies, that have reported a prevalence
of S. aureus in the community around 29-32% (Mainous et al., 2006; Gorwitz et al., 2008) and
among healthcare workers and patients between 29-79% (Kumar et al., 2011; Al-Talib et al.,
2013). S. aureus strains isolated from healthy people did not harbor the mecA and PVL genes,
representing a low risk of transmission in the community. Other studies have reported a nasal
carriage of MRSA approximately of 0.8-1.5% in the community (Mainous et al., 2006;
Gorwitz et al., 2008), 0.5-44% in patients (Tiemersma et al., 2004), 20% in healthcare workers
(Kumar et al., 2011) and 30% in people living and working in farms with MRSA-positive pigs
or dust (Van den Broek et al., 2009). Studies that reported a high prevalence of S. aureus and
MRSA in the community have considered a higher sample size of subjects, with different
demographic characteristics (age, race/ethnicity, sex, place of birth) and with samplings in
different years. In addition, the high prevalence of S. aureus and MRSA among healthcare
workers and patients is due to the exposure to hospital environment with major risks in
transmitting and spreading nosocomial infections.
3.5 Conclusion
Staphylococcus aureus harboring mecA and PVL genes is present in patients affected by
invasive infections. The nasal carriage of S. aureus in healthy humans seems to be low, and
the mecA and PVL genes could not be present. Therefore, MRSA might not represent a severe
risk for transmission among the community.
Further molecular typing techniques are needed to improve the molecular characterization of
the S. aureus isolates obtained from humans.
79
Acknowledgements
This study was supported by the Dean’s Office, College of Agriculture, Food Systems and
Natural Resources College, North Dakota State University (Valeria Velasco), the Dean’s
Office, College of Veterinary Medicine, Iowa State University, Ames, Iowa; and the
Commission of Scientific Research Projects, Uludag University (Project YDP (V)-2009/4).
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84
Table 1 (Desarrollo 3). Nucleotide sequence of the primers used in the multiplex PCR for
detection of the 16S rRNA gene, the mecAgene and the PVL genes (McClure et al., 2006).
Primer Oligonucleotide sequence Amplicon
Size (bp)
Staphy 756 F 5’-AAC TCT GTT ATT AGG GAA GAA CA-3’ Staph 750 R 5’-CCA CCT TCC TCC GGT TTG TCA CC-3’
756
mecA-1 F 5’-GTA GAA ATG ACT GAA CGT CCG ATA A-3’ mecA-2 R 5’-CCA ATT CCA CAT TGT TTC GGT CTA A-3’ 310
luk-PV-1 F 5’-ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A-3’ luk-PV-2 R 5’-GCA TCA AGT GTA TTG GAT AGC AAA AGC-3’ 433
85
Table 2 (Desarrollo 3). Identification of 16S rRNA, mecA and Panton-Valentine Leukocidin
(PVL) genes in Staphylococcus aureus isolates from healthy people and methicillin-resistance
Staphylococcus aureus (MRSA) isolates from hospital patients.
No. of isolates with the specific
gene (%) Source
No. of
samples
No. of samples
positive for S.
aureus (%)
No. of
samples
positive for
MRSA (%)
16S
rRNA mecA PVL
Hospital patients
Blood 99 99 (100) 99 (100) 99 (100) 96 (97.0) 11 (11.1)
Wound 9 9 (100) 9 (100) 9 (100) 9 (100) 0 (0.0)
Total 108 108 (100) 108 (100) 108 (100) 105 (97.2) 11 (10.2)
Healthy people
Fall 2010 231 17 (7.4) 17 (7.4) 0 (0.0) 0 (0.0)
Spring 2011 219 25 (7.8) 25 (7.8) 0 (0.0) 0 (0.0)
Total 550 42 (7.6) 42 (7.6) 0 (0.0) 0 (0.0)
86
III. DISCUSIÓN GENERAL
En este estudio se encontró una alta prevalencia de S. aureus en animales y carne cruda
(35% y 48%, respectivamente), observándos una mayor prevalencia en corderos y cerdos y en
las carnes de pollo y cerdo. Sin embargo, en productos cárnicos la prevalencia de S. aureus fue
menor (13%). En personas sanas, se obtuvo una baja proporción de portadores de S. aureus
(8%), cuando se compara con los resultados de otros estudios (Mainous et al., 2006; Gorwitz
et al., 2008). Adicionalmente, se detectó la presencia del gen mecA sólo en carne de cerdo, con
una baja prevalencia (3%) similar a lo reportado en otras investigaciones (de Boer et al., 2009;
Pu et al., 2009). Finalmente, se determinó en animales (cerdos y corderos) y carne de cerdo la
presencia de S. aureus MR y S. aureus ST398.
La presencia de SARM, S. aureus MR y S. aureus ST398 en animales de abasto, en
carne y productos cárnicos, sugiere que las personas podrían estar expuestas a adquirir este
tipo de agentes de infección a través de la cadena productiva de la carne, existiendo un riesgo
mayor cuando se trata de productos cárnicos que no requieren cocción previa al consumo.
En este estudio no se detectaron los genes PVL en ninguna de las cepas de S. aureus
aisladas de animales, carne y personas sanas. Sólo algunas cepas de SARM obtenidas de
pacientes enfermos hospitalizados (10%) resultaron ser positivas para estos genes de
virulencia.
Los resultados de prevalencia de S. aureus obtenidos en animales y carne mediante el
método de cultivo coincidieron con los resultados obtenidos a través de la técnica de PCR
(identificación del gen ARNr 16S). Por esta razón, resulta conveniente incluir una etapa de
enriquecimiento selectivo previo a la etapa de cultivo selectivo, seguido de la confirmación de
las cepas presuntivas de S. aureus a través de pruebas bioquímicas.
En este estudio la detección de SARM se determinó a través de la técnica de PCR
(identificación del gen mecA) y test de susceptibilidad antimicrobiana (resistencia a
antibióticos β-lactámicos). Existen otros métodos que se han utilizado para confirmar la
presencia de SARM. Estos métodos incluyen tests de susceptibilidad antimicrobiana a la
oxacilina o cefoxitina (Danial et al., 2011; Kumar et al., 2011; Kim et al., 2013; Nimmo et al.,
87
2013) o detección de la proteína PBP2a a través de tests de aglutinación (Anderson and Weese,
2007; Weese et al., 2010; Danial et al., 2011).
El método utilizado para aislar S. aureus y SARM puede influir en los resultados de
prevalencia. Algunos autores han utilizado sólo una etapa de cultivo en placa (Weese et al.,
2006; Aydin et al., 2011), y otros han incluido etapas de enriquecimiento selectivo previo a la
etapa de cultivo (Wertheim et al., 2001; de Boers et al., 2009; Pu et al., 2009; Tenhagen et al.,
2009; Broens et al., 2011; Pu et al., 2011; Zhang et al., 2011). de Boer et al. (2009) utilizando
las etapas de enriquecimiento primario y secundario, como se describe en el presente trabajo,
obtuvieron una mayor detección de SARM en muestras de carne. Tanto las cepas de S. aureus
y SARM pueden ser aisladas utilizando medios selectivos que contienen NaCl. Sin embargo,
algunas cepas de SARM no crecen cuando la concentración excede de 2,5% (Jones et al.,
1997). Además, algunos medios suplementados con antibióticos podrían causar un crecimiento
exacerbado de cepas de S. aureus susceptibles a la meticilina (Böcher et al., 2008) o fallar en
el aislamiento de SARM. Por esta razón, se recomienda incluir siempre un medio de cultivo
libre de antibióticos, aunque el objetivo sea la identificación de SARM (Pu et al., 2009).
La rápida detección de S. aureus y SARM en animales y carne permitiría disminuir el
riesgo de exposición a contaminación en la cadena productiva de la carne. En este estudio para
disminuir los tiempos de detección, se desarrolló un ensayo de PCR múltiple en tiempo real
con el fin de detectar los genes nuc (identificar S. aureus), mecA (asociado a SARM) y PVL
(factor de virulencia), en muestras extraídas de animales y carne. Se encontró una
concordancia total alta entre el método de cultivo convencional y la técnica de PCR en tiempo
real, con una mayor detección al utilizar el medio de enriquecimiento secundario. Lo anterior
se debería a la recuperación de células dañadas al utilizar el enriquecimiento secundario y a
que niveles bajos de S. aureus no serían detectados al utilizar sólo el enriquecimiento primario
en PCR en tiempo real. Sin embargo, la técnica de cultivo convencional sigue siendo
considerada la metodología estándar para la identificación de S. aureus (Huletsky et al., 2004;
Paule et al., 2005; Danial et al., 2011) y PCR convencional (detección del gen mecA) para la
identificación de SARM (Maes et al., 2002; Makgotlho et al., 2009).
88
Para la técnica de PCR en tiempo real, se utilizó ADN extraído de los caldos de
enriquecimiento de las muestras obtenidas de animales y carne, por lo cual la concentración y
procedencia del material genético fue variable. Se detectó la presencia del gen mecA en
muestras que resultaron ser negativas para S. aureus, pero positivas para Staphylococcus spp. a
través del método de cultivo y test bioquímicos. Estos resultados son considerados falsos
positivos, ya que se ha detectado el gen mecA tanto en S. aureus como en cepas staphylococci
coagulasa-negativa (Ryffel et al., 1990; Hagen et al., 2005; Higashide et al., 2006; Thomas et
al., 2007; Black et al., 2011). Desafortunadamente, en este estudio los resultados no pudieron
ser validados a través del método de cultivo, ya que se utilizó extractos de ADN, habiéndose
inactivado las células previamente.
El método de PCR en tiempo real utilizado en este estudio debió evaluarse en términos
de sensibilidad de detección, determinando la concentración mínima de ADN para obtener
amplificación. Además de utilizar las muestras control externo, se recomienda incorporar un
control interno de amplificación que podría indicar resultados falsos-negativos, por la
presencia de inhibidores en la reacción, mal funcionamiento del termociclador, actividad baja
de la polimerasa o incorrecta solución de PCR (Hoorfar et al., 2004).
La similitud genética encontrada entre las cepas de S. aureus aisladas de cerdos y carne
de cerdo (ST9), sugiere la posible contaminación de la carne durante la etapa de la faena. De
las cepas positivas para el gen mecA, dos resultaron ser ST5, y tres ST398, todas encontradas
en carne de cerdo. Además, se encontró una alta prevalencia de S. aureus ST398 (no-SARM),
lo cual indica un riesgo potencial para las personas de adquirir esta infección a través de la
cadena productiva de la carne.
Para la tipificación molecular de S. aureus a través de PFGE se sigue el protocolo de
PulseNet, que utiliza la enzima de restricción SmaI. Sin embargo, esta enzima no puede digerir
el ADN de las cepas ST398, debido a que presentan una enzima que metila la secuencia de
reconocimiento de SmaI (Bens et al., 2006). Por lo tanto, no es posible obtener un patrón de
restricción con PFGE para ser comparado con otras cepas. Por esta razón, en este estudio se
utilizó una segunda enzima de restricción, XmaI (un isoesquizómero de SmaI), con la cual se
obtuvo patrones de restricción con bandas muy débiles de las cepas ST398. Para poder realizar
89
un análisis más preciso de la tipificación molecular de S. aureus ST398 con PFGE, se debe
buscar enzimas de restricción que actúen sobre otras secuencias de reconocimiento, diferentes
a las enzimas SmaI y XmaI, y así establecer un protocolo de PFGE para las cepas que no
presenten un patrón de restricción definido.
Para tener mayor precisión en la tipificación molecular de las cepas, se recomienda la
utilización de al menos dos técnicas moleculares (Tenover et al., 1994). En este estudio se
utilizó la técnica PFGE y MLST en la tipificación de las cepas de S. aureus. Generalmente,
PFGE y MLST clasifican las cepas en clusters similares (Catry et al., 2010). Otro método de
tipificación utilizado para S. aureus es la tipificación de spa, la cual tiene como desventaja de
que líneas clonales no relacionadas pueden tener spa tipos similares (Van den Broek IV et al.,
2009; Golding et al., 2008). Por esta razón, se podrían obtener discrepancias con respecto a los
resultados a través de MLST y PFGE (Golding et al., 2008). Sin embargo, el método de
tipificación molecular con mayor poder de discriminación es PFGE (McDougal et al., 2003).
Todas las cepas SARM detectadas en este estudio resultaron ser resistentes a la
penicilina a través del test de susceptibilidad antimicrobiana, obteniéndose una CMI mayor
que en las otras cepas de S. aureus. Además, se obtuvo una alta prevalencia de S. aureus MR
[resistente al menos a tres agentes antimicrobianos (Aydin et al., 2011)] en animales y en
carne cruda, principalmente a los antibióticos penicilina, tetraciclina, lincomicina y
eritromicina. La multirresistencia se presentó principalmente en cepas ST398 y ST9, las cuales
se asocian a animales, específicamente a cerdos (Lewis et al., 2008; van Belkum et al., 2008;
Guardabassi et al., 2009; Krziwanek et al., 2009).
Los agentes antimicrobianos son drogas efectivas contra los agentes infecciones. Sin
embargo, su uso imprudente contribuye a la resistencia bacteriana, ya sea en hospitales, en la
comunidad o en producción animal. El uso extensivo de agentes antimicrobianos ejerce una
presión selectiva sobre las cepas resistentes, eliminando los competidores susceptibles (Swartz,
1997; Marinelli y Tomasz, 2010). Los glicopéptidos, como la vancomicina, son
frecuentemente utilizados para el tratamiento de infecciones causadas por SARM, sin embargo,
en los últimos años ha aumentado la incidencia de S. aureus con resistencia y resistencia
intermedia a este antibiótico (Tiwari y Sen, 2006). En este estudio todas las cepas de S. aureus
90
aisladas de animales y carne fueron susceptibles a vancomicina, así como también a
daptomicina, linezolida y nitrofurantoina, coincidiendo con los resultados obtenidos por Pu et
al. (2011) en muestras de carne. Los estudios anteriores y los resultados de este estudio
sugieren que las infecciones por S. aureus y SARM originarios de animales portadores o de
carne contaminada podrían ser tratadas con daptomicina, linezolida o nitrofurantoina por la
susceptibilidad que presentan las cepas.
En producción animal, los antibióticos se utilizan como profilácticos, en el tratamiento
de enfermedades y como promotores del crecimiento (DuPont y Steele, 1987; Franco et al.,
1990). Esto puede generar patógenos resistentes en animales, existiendo el riesgo potencial de
que los genes de resistencia sean incorporados en bacterias presentes en humanos. En este
estudio, la identificación de S. aureus MR en animales y en carne plantea el riesgo de
transmisión a las personas a través de la cadena de la carne y explicaría la disminución de la
eficacia de este tipo de medicamentos utilizados en salud humana (Smith et al., 2002).
Se recomienda que los hospitales disminuyan la utilización de un antibiótico antes de
que las cepas resistentes se propaguen. Sin embargo, puede ocurrir que el gen de resistencia
haya sido transpasado a plásmidos que portan resistencia a agentes antimicrobianos utilizados
como alternativas de la droga original (Swartz, 1997). Se debe aumentar los esfuerzos para
reducir el uso de antibióticos, asegurar el control de las infecciones y los mecanismos de
vigilancia, y seleccionar las dosis y combinaciones utilizadas, para evitar la emergencia de
resistencia (Marinelli y Tomasz, 2010).
En la actualidad se están desarrollando nuevos agentes antimicrobianos que sean
efectivos contra los patógenos resistentes. Las investigaciones que se desarrolllen en este
ámbito debieran considerar la evaluación de la susceptibilidad de S. aureus a estos nuevos
antimicrobianos y combinaciones de ellos y, además, investigar aún más acerca de los
diferentes mecanismos de resistencia que pueden presentarse en los microorganismos. De esta
forma, se podrá contar con la información necesaria para poder controlar S. aureus resistente y
disminuir la exposición de las personas en las diferentes etapas de la cadena productiva de la
carne.
91
En Estados Unidos existe un sistema de vigilancia llamado National Antimicrobial
Resistance Monitoring System (NARMS), creado en 1996 en colaboración con FDA (Food
and Drug Administration), CDC, USDA (U.S. Department of Agriculture) y los
departamentos de salud estatales y locales. NARMS realiza un monitoreo de la susceptibilidad
antimicrobiana de bacterias entéricas aisladas en humanos, carne y animales de producción.
Los patógenos que se incluyen en NARMS son Salmonella spp., Escherichia coli,
Campylobacter spp., Shigella spp. y Enterococcus spp. (FDA, 2012). Sin embargo, la
evidencia que existe de la presencia de SARM y S. aureus MR en la cadena productiva de la
carne, sugiere que se debiera ampliar el monitoreo a otros patógenos resistentes, no sólo las
bacterias entéricas que se incluyen actualmente. De esta forma, al incluir S. aureus en este
sistema nacional de monitoreo se podría conocer año a año la prevalencia de las cepas
resistentes, la susceptibilidad antimicrobiana que presentan, evaluar la susceptibilidad a
nuevos agentes antimicrobianos y establecer estrategias de control y mitigación para disminuir
la exposición de las personas.
En este estudio se analizaron cepas SARM obtenidas de pacientes enfermos, de las
cuales tres fueron negativas para el gen mecA. Esto podría deberse a la obtención de resultados
falsos positivos en el hospital o a la presencia de un nuevo homólogo del gen mecA,
recientemente identificado como mecC, el cual ha sido detectado en SARM negativo para el
gen mecA, aislado de humanos y animales (García-Álvarez et al., 2011; Ito et al., 2012;
Laurent et al., 2012; Monecke et al., 2013; Petersen et al., 2013). Además, se extrajeron
muestras de personas sanas para determinar la proporción de portadores de SARM sanos,
resultando ser negativo para todos los individuos. Para obtener una mayor precisión en la
identificación y caracterización de SARM en estas muestras, se recomienda la detección de
ambos genes (mecA y mecC), además de la determinación de la susceptibilidad antimicrobiana,
y tipificación molecular a través de PFGE y MLST.
92
IV. CONCLUSIONES GENERALES
De los resultados obtenidos en este estudio se puede concluir que:
Existe una alta prevalencia de S. aureus en animales productores de carne y carne
cruda y una baja proporción de personas portadoras sanas. Las cepas de S. aureus aisladas de
animales, carne y personas sanas en este estudio no son portadoras de los genes PVL. Cepas
de S. aureus extraídas de pacientes enfermos que padecen infecciones presentan los genes
mecA, asociado a la resistencia y PVL, asociado a la virulencia. A pesar de que la prevalencia
de SARM en carne cruda es baja, la alta prevalencia de S. aureus MR, y cepas ST398 en la
cadena productiva de la carne, sugiere que existe un riesgo de transmisión a las personas.
Además, la similitud genética entre las cepas de S. aureus encontradas en animales y carne,
sugiere la contaminación de la carne durante la faena.
Se recomienda incorporar la detección de SARM y S. aureus MR y la determinación
de la susceptibilidad antimicrobiana de las cepas en los sistemas de vigilancia que existen a
nivel federal, como es NARMS (National Antimicrobial Resistence Monitoring System),
considerando el monitoreo de la cadena de la carne, de pacientes en los hospitales y de
personas sanas en la comunidad.
La utilización de medios de enriquecimiento selectivos previo a la etapa de cultivo
permite aislar S. aureus y SARM, obteniéndose 100% de concordancia con la técnica de PCR
convencional. La utilización de un medio de enriquecimiento secundario junto con PCR en
tiempo real mejora la detección de S. aureus en muestras de animales y carne. La técnica de
PCR en tiempo real permite una detección rápida de S. aureus y el gen mecA, siendo
recomendable confirmar la presencia de SARM a través de la metodología estándar.
Para establecer medidas de control de la propagación de las cepas resistentes de S.
aureus en la cadena productiva de la carne es necesario determinar la tipificación molecular y
la susceptibilidad antimicrobiana.
93
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